US20030190644A1 - Methods for generating databases and databases for identifying polymorphic genetic markers - Google Patents

Methods for generating databases and databases for identifying polymorphic genetic markers Download PDF

Info

Publication number
US20030190644A1
US20030190644A1 US10/272,756 US27275602A US2003190644A1 US 20030190644 A1 US20030190644 A1 US 20030190644A1 US 27275602 A US27275602 A US 27275602A US 2003190644 A1 US2003190644 A1 US 2003190644A1
Authority
US
United States
Prior art keywords
dna
data
population
sequence
samples
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/272,756
Inventor
Andreas Braun
Yip Ping
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sequenom Inc
Original Assignee
Sequenom Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/663,968 external-priority patent/US7917301B1/en
Application filed by Sequenom Inc filed Critical Sequenom Inc
Priority to US10/272,756 priority Critical patent/US20030190644A1/en
Assigned to SEQUENOM, INC. reassignment SEQUENOM, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YIP, PING, CHIU, NORMAN, KOSTER, HUBERT, RODI, CHARLIE, VAN DEN BOOM, DIRK, BRAUN, ANDREAS, HE, LIYAN, JURINKE, CHRISTIAN
Publication of US20030190644A1 publication Critical patent/US20030190644A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing
    • C12Q1/6872Methods for sequencing involving mass spectrometry
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6827Hybridisation assays for detection of mutation or polymorphism
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/24Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

  • SNPs single nucleotide polymorphisms
  • microsatellites tandem repeats
  • tandem repeats newly mapped introns and exons
  • SNPs Single Nucleotide Polymorphisms
  • the databases comprise sets of parameters associated with subjects in populations selected only on the basis of being healthy (i e., where the subjects are mammals, such as humans, they are selected based upon apparent health and no detectable infections).
  • the databases can be sorted based upon one or more of the selected parameters.
  • the databases can be relational databases, in which an index that represents each subject serves to relate parameters, which are the data, such as age, ethnicity, sex, medical history, etc. and ultimately genotypic information, that was inputted into and stored in the database.
  • the database can then be sorted according to these parameters. Initially, the parameter information is obtained from a questionnaire answered by each subject from whom a body tissue or body fluid sample is obtained. As additional information about each sample is obtained, this information can be entered into the database and can serve as a sorting parameter.
  • the databases obtained from healthy individuals have numerous uses, such as correlating known polymorphisms with a phenotype or disease.
  • the databases can be used to identify alleles that are deleterious, that are beneficial, and that are correlated with diseases.
  • genotypic information can be obtained by any method known to those of skill in the art, but is generally obtained using mass spectrometry.
  • any database can be sorted according to the methods herein, and alleles that exhibit statistically significant correlations with any of the sorting parameters can be identified. It is noted, however, is noted, that the databases provided herein and randomly selected databases will perform better in these methods, since disease-based databases suffer numerous limitations, including their relatively small size, the homogeneity of the selected disease population, and the masking effect of the polymorphism associated with the markers for which the database was selected. Hence, the healthy database provided herein, provides advantages not heretofore recognized or exploited.
  • the methods provided herein can be used with a selected database, including disease-based databases, with or without sorting for the discovery and correlation of polymorphisms.
  • the databases provided herein represent a greater genetic diversity than the unselected databases typically utilized for the discovery of polymorphisms and thus allow for the enhanced discovery and correlation of polymorphisms.
  • the databases provided herein can be used for taking an identified polymorphism and ascertaining whether it changes in frequency when the data are sorted according to a selected parameter.
  • One use of these methods is correlating a selected marker with a particular parameter by following the occurrence of known genetic markers and then, having made this correlation, determining or identifying correlations with diseases. Examples of this use are p53 and Lipoprotein Lipase polymorphism. As exemplified herein, known markers are shown to have particular correlation with certain groups, such as a particular ethnicity or race or one sex. Such correlations will then permit development of better diagnostic tests and treatment regimens.
  • the databases and methods provided herein permit, among other things, identification of components, particularly key components, of a disease process by understanding its genetic underpinnings and also permit an understanding of processes, such as individual drug responses.
  • the databases and methods provided herein also can be used in methods involving elucidation of pathological pathways, in developing new diagnostic assays, identifying new potential drug targets, and in identifying new drug candidates.
  • the methods and databases can be used with experimental procedures, including, but are not limited to, in silico SNP identification, in vitro SNP identification/verification, genetic profiling of large populations, and in biostatistical analyses and interpretations.
  • kits that contain a database provided herein and a biological sample from a subject in the database, and typically biological samples from all subjects or a plurality of subjects in the database. Collections of the tissue and body fluid samples are also provided.
  • determining whether a genetic marker correlates with susceptibility to morbidity, early mortality, or morbidity and early mortality comprising identifying a polymorphism and determining the frequency of the polymorphism with increasing age in a healthy population.
  • the method and system for identifying a biological sample generates a data set indicative of the composition of the biological sample.
  • the data set is DNA spectrometry data received from a mass spectrometer.
  • the data set is denoised, and a baseline is deleted. Since possible compositions of the biological sample can be known, expected peak areas can be determined. Using the expected peak areas, a residual baseline is generated to further correct the data set. Probable peaks are then identifiable in the corrected data set, which are used to identify the composition of the biological sample.
  • statistical methods are employed to determine the probability that a probable peak is an actual peak, not an actual peak, or that the data too inconclusive to call.
  • the method and system for identifying a biological sample accurately makes composition calls in a highly automated manner.
  • complete SNP profile information for example, can be collected efficiently.
  • the collected data are analyzed with highly accurate results. For example, when a particular composition is called, the result can be relied upon with great confidence.
  • Such confidence is provided by the robust computational process employed
  • FIG. 1 depicts an exemplary sample bank.
  • Panel 1 shows the samples as a function of sex and ethnicity.
  • Panel 2 shows the Caucasians as a function of age.
  • Panel 3 shows the Hispanics as a function of age.
  • FIGS. 2A and 2C show an age- and sex-distribution of the 291S allele of the lipoprotein lipase gene in which a total of 436 males and 589 females were investigated.
  • FIG. 2B shows an age distribution for the 436 males.
  • FIG. 3 is an exemplary questionnaire for population-based sample banking.
  • FIG. 4 depicts processing and tracking of blood sample components.
  • FIG. 5 depicts the allelic frequency of “sick” alleles and “healthy” alleles as a function of age. It is noted that the relative frequency of healthy alleles increases in a population with increasing age.
  • FIG. 6 depicts the age-dependent distribution of ApoE genotypes (see, Schchter et al. (1994) Nature Genetics 6:29-32).
  • FIG. 7A-D depicts age-related and genotype frequency of the p53 (tumor suppressor) codon 72 among the Caucasian population in the database.
  • *R72 and *P72 represent the frequency of the allele in the database population.
  • R72, R72P, and P72 represent the genotypes of the individuals in the population.
  • the frequency of the homozygous P72 allele drops from 6.7% to 3.7% with age.
  • FIG. 8 depicts the allele and genotype frequencies of the p21 S31R allele as a function of age.
  • FIG. 9 depicts the frequency of the FVII Allele 353Q in pooled versus individual samples.
  • FIG. 10 depicts the frequency of the CETP (cholesterol ester transfer protein) allele in pooled versus individual samples.
  • FIG. 11 depicts the frequency of the plasminogen activator inhibitor-1 (PAI-1) 5G in pooled versus individual samples.
  • FIG. 12 shows mass spectra of the samples and the ethnic diversity of the PAI-1 alleles.
  • FIG. 13 shows mass spectra of the samples and the ethnic diversity of the CETP 405 alleles.
  • FIG. 14 shows mass spectra of the samples and the ethnic diversity of the Factor VII 353 alleles.
  • FIG. 15 shows ethnic diversity of PAI-1, CETP and Factor VII using the pooled DNA samples.
  • FIG. 16 shows the p53-Rb pathway and the relationships among the various factors in the pathway.
  • FIG. 17 which is a block diagram of a computer constructed to provide and process the databases described herein, depicts a typical computer system for storing and sorting the databases provided herein and practicing the methods provided herein.
  • FIG. 18 is a flow diagram that illustrates the processing steps performed using the computer illustrated in FIG. 17, to maintain and provide access to the databases for identifying polymorphic genetic markers.
  • FIG. 19 is a histogram showing the allele and genotype distribution in the age and sex stratified Caucasian population for the AKAP10-1 locus. Bright green bars show frequencies in individuals younger than 40 years. Dark green bars show frequencies in individuals older than 60 years.
  • FIG. 20 is a histogram showing the allele and genotype distribution in the age and sex stratified Caucasian population for the AKAP10-5 locus. Bright green bars show frequencies in individuals younger than 40 years; dark green bars show frequencies in individuals older than 60 years.
  • FIG. 21 is a histogram showing the allele and genotype distribution in the age and sex stratified Caucasian population for the h-msrA locus. Genotype difference between male age groups is significant. Bright green bars show frequencies in individuals younger than 40 years. Dark green bars show frequencies in individuals older than 60 years.
  • FIG. 22A-D is a sample data collection questionnaire used for the healthy database.
  • FIG. 23 is a flowchart showing processing performed by the computing device of FIG. 24 when performing genotyping of sense strands and antisense strands from assay fragments.
  • FIG. 24 is a block diagram showing a system provided herein;
  • FIG. 25 is a flowchart of a method of identifying a biological sample provided herein;
  • FIG. 26 is a graphical representation of data from a mass spectrometer
  • FIG. 27 is a diagram of wavelet transformation of mass spectrometry data
  • FIG. 28 is a graphical representation of wavelet stage 0 hi data
  • FIG. 29 is a graphical representation of stage 0 noise profile
  • FIG. 30 is a graphical representation of generating stage noise standard deviations
  • FIG. 31 is a graphical representation of applying a threshold to data stages
  • FIG. 32 is a graphical representation of a sparse data set
  • FIG. 33 is a formula for signal shifting
  • FIG. 34 is a graphical representation of a wavelet transformation of a denoised and shifted signal
  • FIG. 35 is a graphical representation of a denoised and shifted signal
  • FIG. 36 is a graphical representation of removing peak sections
  • FIG. 37 is a graphical representation of generating a peak free signal
  • FIG. 38 is a block diagram of a method of generating a baseline correction
  • FIG. 39 is a graphical representation of a baseline and signal
  • FIG. 40 is a graphical representation of a signal with baseline removed
  • FIG. 41 is a table showing compressed data
  • FIG. 42 is a flowchart of method for compressing data
  • FIG. 43 is a graphical representation of mass shifting
  • FIG. 44 is a graphical representation of determining peak width
  • FIG. 45 is a graphical representation of removing peaks
  • FIG. 46 is a graphical representation of a signal with peaks removed
  • FIG. 47 is a graphical representation of a residual baseline
  • FIG. 48 is a graphical representation of a signal with residual baseline removed
  • FIG. 49 is a graphical representation of determining peak height
  • FIG. 50 is a graphical representation of determining signal-to-noise for each peak
  • FIG. 51 is a graphical representation of determining a residual error for each peak
  • FIG. 52 is a graphical representation of peak probabilities
  • FIG. 53 is a graphical representation of applying an allelic ratio to peak probability
  • FIG. 54 is a graphical representation of determining peak probability
  • FIG. 55 is a graphical representation of calling a genotype
  • FIG. 56 is a flowchart showing a statistical procedure for calling a genotype
  • FIG. 57 is a flowchart showing processing performed by the computing device of FIG. 1 when performing standardless genotyping.
  • FIG. 58 is graphical representation of applying an allelic ratio to peak probability for standardless genotype processing.
  • a biopolymer includes, but is not limited to, nucleic acid, proteins, polysaccharides, lipids and other macromolecules.
  • Nucleic acids include DNA, RNA, and fragments thereof. Nucleic acids can be derived from genomic DNA, RNA, mitochondrial nucleic acid, chloroplast nucleic acid and other organelles with separate genetic material.
  • morbidity refers to conditions, such as diseases or disorders, that compromise the health and well-being of an organism, such as an animal.
  • Morbidity susceptibility or morbidity-associated genes are genes that, when altered, for example, by a variation in nucleotide sequence, facilitate the expression of a specific disease clinical phenotype.
  • morbidity susceptibility genes have the potential, upon alteration, of increasing the likelihood or general risk that an organism will develop a specific disease.
  • mortality refers to the statistical likelihood that an organism, particularly an animal, will not survive a full predicted lifespan. Hence, a trait or a marker, such as a polymorphism, associated with increased mortality is observed at a lower frequency in older than younger segments of a population.
  • a polymorphism refers to a variation in the sequence of a gene in the genome amongst a population, such as allelic variations and other variations that arise or are observed.
  • a polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. These differences can occur in coding and non-coding portions of the genome, and can be manifested or detected as differences in nucleic acid sequences, gene expression, including, for example transcription, processing, translation, transport, protein processing, trafficking, DNA synthesis, expressed proteins, other gene products or products of biochemical pathways or in post-translational modifications and any other differences manifested amongst members of a population.
  • a single nucleotide polymorphism refers to a polymorphism that arises as the result of a single base change, such as an insertion, deletion or change in a base.
  • a polymorphic marker or site is the locus at which divergence occurs. Such site can be as small as one base pair (an SNP).
  • Polymorphic markers include, but are not limited to, restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats and other repeating patterns, simple sequence repeats and insertional elements, such as Alu.
  • Polymorphic forms also are manifested as different mendelian alleles for a gene. Polymorphisms can be observed by differences in proteins, protein modifications, RNA expression modification, DNA and RNA methylation, regulatory factors that alter gene expression and DNA replication, and any other manifestation of alterations in genomic nucleic acid or organelle nucleic acids.
  • a healthy population refers to a population of organisms, including but are not limited to, animals, bacteria, viruses, parasites, plants, eubacteria, and others, that are disease free.
  • the concept of disease-free is a function of the selected organism. For example, for mammals it refers to a subject not manifesting any disease state.
  • Practically a healthy subject, when human, is defined as human donor who passes blood bank criteria to donate blood for eventual use in the general population. These criteria are as follows: free of detectable viral, bacterial, mycoplasma, and parasitic infections; not anemic; and then further selected based upon a questionnaire regarding history (see FIG. 3).
  • a healthy population represents an unbiased population of sufficient health to donate blood according to blood bank criteria, and not further selected for any disease state.
  • individuals are not taking any medications.
  • plants for example, it is a plant population that does not manifest diseases pathology associated with plants.
  • bacteria it is a bacterial population replicating without environmental stress, such as selective agents, heat and other pathogens.
  • a healthy database refers to a database of profiles of subjects that have not been pre-selected for any particular disease. Hence, the subjects that serve as the source of data for the database are selected, according to predetermined criteria, to be healthy. In contrast to other such databases that have been pre-selected for subjects with a particular disease or other characteristic, the subjects for the database provided herein are not so-selected. Also, if the subjects do manifest a disease or other condition, any polymorphism discovered or characterized should be related to an independent disease or condition. In a one embodiment, where the subjects are human, a healthy subject manifests no disease symptoms and meets criteria, such as those set by blood banks for blood donors.
  • the subjects for the database are a population of any organism, including, but are not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity that has nucleic acid.
  • subjects are mammals, such as, although not necessarily, humans.
  • Such a database can capture the diversity of a population, thus providing for discovery of rare polymorphisms.
  • a profile refers to information relating to, but not limited to and not necessarily including all of, age, sex, ethnicity, disease history, family history, phenotypic characteristics, such as height and weight and other relevant parameters.
  • a sample collect information form is shown in FIG. 22, which illustrates profile intent.
  • a disease state is a condition or abnormality or disorder that can be inherited or result from environmental stresses, such as toxins, bacterial, fungal and viral infections.
  • set of non-selected subjects means that the subjects have not been pre-selected to share a common disease or other characteristic. They can be selected to be healthy as defined herein.
  • a phenotype refers to a set of parameters that includes any distinguishable trait of an organism.
  • a phenotype can be physical traits and can be, in instances in which the subject is an animal, a mental trait, such as emotional traits. Some phenotypes can be determined by observation elicited by questionnaires (see, e.g., FIGS. 3 and 22) or by referring to prior medical and other records.
  • a phenotype is a parameter around which the database can be sorted.
  • a parameter is any input data that will serve as a basis for sorting the database. These parameters will include phenotypic traits, medical histories, family histories and any other such information elicited from a subject or observed about the subject. A parameter can describe the subject, some historical or current environmental or social influence experienced by the subject, or a condition or environmental influence on someone related to the subject. Paramaters include, but are not limited to, any of those described herein, and known to those of skill in the art.
  • haplotype refers to two or polymorphism located on a single DNA strand.
  • haplotyping refers to identification of two or more polymorphisms on a single DNA strand.
  • Haplotypes can be indicative of a phenotype. For some disorders a single polymorphism can suffice to indicate a trait; for others a plurality (i.e., a haplotype) can be needed.
  • Haplotyping can be performed by isolating nucleic acid and separating the strands. In addition, when using enzymes such a certain nucleases, that produce, different size fragments from each strand, strand separation is not needed for haplotyping.
  • pattern with reference to a mass spectrum or mass spectrometric analyses refers to a characteristic distribution and number of signals (such peaks or digital representations thereof).
  • signal in the context of a mass spectrum and analysis thereof refers to the output data, which the number or relative number of moleucles having a particular mass. Signals include “peaks” and digital representations thereof.
  • adaptor when used with reference to haplotyping using Fen ligase, refers to a nucleic acid that specifically hybridizes to a polymorphism of interest.
  • An adaptor can be partially double-stranded.
  • An adaptor complex is formed when an adaptor hybridizes to its target.
  • a target nucleic acid refers to any nucleic acid of interest in a sample. It can contain one or more nucleotides.
  • standardless analysis refers to a determination based upon an internal standard. For example, the frequency of a polymorphism can be determined herein by comparing signals within a single mass spectrum.
  • amplifying refers to methods for increasing the amount of a bipolymer, especially nucleic acids. Based on the 5′ and 3′ primers that are chosen, amplication also serves to restrict and define the region of the genome which is subject to analysis. Amplification can be performed by any method known to those skilled in the art, including use of the polymerase chain reaction (PCR) etc. Amplification, e.g., PCR must be done quantitatively when the frequency of polymorphism is required to be determined.
  • PCR polymerase chain reaction
  • cleaving refers to non-specific and specific fragmentation of a biopolymer.
  • multiplexing refers to the simultaneous detection of more than one polymorphism.
  • Methods for performing multiplexed reactions, particularly in conjunction with mass spectrometry are known (see, e.g., U.S. Pat. Nos. 6,043,031, 5,547,835 and International PCT application No. WO 97/37041).
  • mass spectrometry encompasss any suitable mass spectrometric format known to those of skill in the art.
  • Such formats iniude, but are not limited to, Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF), Electrospray (ES), IR-MALDI (see, e.g., published International PCT application No.99/57318 and U.S. Pat. No. 5,118,937), Ion Cyclotron Resonance (ICR), Fourier Transform and combinations thereof.
  • MALDI particular UV and IR, are among the formats contemplated.
  • mass spectrum refers to the presentation of data obtained from analyzing a biopolymer or fragment thereof by mass spectrometry either graphically or encoded numerically.
  • a blood component is a component that is separated from blood and includes, but is not limited to red blood cells and platelets, blood clotting factors, plasma, enzymes, plasminogen, immunoglobulins.
  • a cellular blood component is a component of blood, such as a red blood cell, that is a cell.
  • a blood protein is a protein that is normally found in blood. Examples of such proteins are blood factors VII and VII. Such proteins and components are well-known to those of skill in the art.
  • plasma can be prepared by any method known to those of skill in the art. For example, it can be prepared by centrifuging blood at a force that pellets the red cells and forms an interface between the red cells and the buffy coat, which contains leukocytes, above which is the plasma.
  • typical platelet concentrates contain at least about 10% plasma.
  • Blood can be separated into its components, including, but not limited to, plasma, platelets and red blood cells by any method known to those of skill in the art. For example, blood can be centrifuged for a sufficient time and at a sufficient acceleration to form a pellet containing the red blood cells. Leukocytes collect primarily at the interface of the pellet and supernatant in the buffy coat region. The supernatant, which contains plasma, platelets, and other blood components, can then be removed and centrifuged at a higher acceleration, whereby the platelets pellet.
  • p53 is a cell cycle control protein that assesses DNA damage and acts as a transcription factor regulation gene which control cell growth, DNA repair and apoptosis.
  • the p53 mutations have been found in a wide variety of different cancers, including all of the different types of leukemia, with varying frequency. The loss of normal p53 functions results in genomic instability and uncontrolled growth of the host cell.
  • p21 is a cyclin-dependent kinase inhibitor, associated with G1 phase arrest of normal cells. Expression triggers apoptosis or programmed cell death and has been associated with Wilms' tumor, a pediatric kidney cancer.
  • Factor VII is a serine protease involved the extrinsic blood coagulation cascade. This factor is activated by thrombin and works with tissue factor (Factor III) in the processing of Factor X to Factor Xa.
  • tissue factor Factor III
  • Evidence has supported an association between polymorphisms in the gene and increase Factor VII activity which can result in an elevated risk of ischemic cardiovascular disease including myocardial infarction.
  • a relational database stores information in a form representative of matrices, such as two-dimensional tables, including rows and columns of data, or higher dimensional matrices.
  • the relational database has separate tables each with a parameter.
  • the tables are linked with a record number, which also acts as an index.
  • the database can be searched or sorted by using data in the tables and is stored in any suitable storage medium, such as floppy disk, CD rom disk, hard drive or other suitable medium.
  • a bar codes refers any array of optically readable marks of any desired size and shape that are arranged in a reference context or frame of, typically, although not necessarily, one or more columns and one or more rows.
  • the bar code refers to any symbology, not necessary “bar” but can include dots, characters or any symbol or symbols.
  • symbology refers to an identifier code or symbol, such as a bar code, that is linked to a sample.
  • the index will reference each such symbology.
  • the symbology is any code known or designed by the user.
  • the symbols are associated with information stored in the database. For example, each sample can be uniquely identified with an encoded symbology.
  • the parameters such as the answers to the questions and subsequent genotypic and other information obtained upon analysis of the samples is included in the database and associated with the symbology.
  • the database is stored on any suitable recording medium, such as a hard drive, a floppy disk, a tape, a CD ROM, a DVD disk and any other suitable medium.
  • first databases of parameters associated with non-selected, particularly healthy, subjects. Also provided are combinations of the databases with indexed samples obtained from each of the subjects. Further provided are databases produced from the first databases. These contain, in addition to the original parameters, information, such as genotypic information, including, but are not limited to, genomic sequence information, derived from the samples.
  • the databases which are herein designated healthy databases, are so-designated because they are not obtained from subjects pre-selected for a particular disease. Hence, although individual members can have a disease, the collection of individuals is not selected to have a particular disease.
  • the subjects from whom the parameters are obtained comprise either a set of subjects who are randomly selected across, typically, all populations, or are pre-selected to be disease-free or healthy.
  • the database is not selected to be representative of any pre-selected phenotype, genotype, disease or other characteristic.
  • the number of subjects from which the database is prepared is selected to produce statistically significant results when used in the methods provided herein.
  • the number of subjects will be greater than 100, 200, and typically than 1000.
  • the precise number can be empirically determined based upon the frequency of the parameter(s) that can be used to sort the database.
  • the population can have at least 50, at least 100, at least 200, at least 500, at least 1000, at least 5000 or at least 10,000 or more subjects.
  • information about each subject is recorded and associated with each subject as a database.
  • the information associated with each of the subjects includes, but is not limited to, information related to historical characteristics of the subjects, phenotypic characteristics and also genotypic characteristics, medical characteristics and any other traits and characteristics about the subject that can be determined. This information will serve as the basis for sorting the database.
  • the subjects are mammals, such as humans, and the information relates to one or more of parameters, such as age, sex, medical history, ethnicity and any other factor.
  • parameters such as age, sex, medical history, ethnicity and any other factor.
  • Such information when the animals are humans, for example, can be obtained by a questionnaire and by observations about the individual, such as hair color, eye color and other characteristics.
  • Genotypic information can be obtained from tissue or other body and body fluid samples from the subject.
  • the healthy genomic database can include profiles and polymorphisms from healthy individuals from a library of blood samples where each sample in the library is an individual and separate blood or other tissue sample. Each sample in the database is profiled as to the sex, age, ethnic group, and disease history of the donor.
  • the databases are generated by first identifying healthy populations of subjects and obtaining information about each subject that will serve as the sorting parameters for the database. This information can be entered into a storage medium, such as the memory of a computer.
  • the information obtained about each subject in a population used for generating the database is stored in a computer memory or other suitable storage medium.
  • the information is linked to an identifier associated with each subject.
  • the database will identify a subject, for example by a datapoint representative of a bar code, and then all information, such as the information from a questionnaire, regarding the individual is associated with the datapoint. As the information is collected the database is generated.
  • profile information such as subject histories obtained from questionnaires
  • the resulting database can be sorted as desired, using standard software, such as by age, sex and/or ethnicity.
  • An exemplary questionnaire for subjects from whom samples are to be obtained is shown in FIGS. 22 A-D.
  • Each questionnaire for example, can be identified by a bar code, particularly a machine readable bar code for entry into the database.
  • the data in the questionnaire is entered into the database and is associated with the bar code.
  • a tissue, cell or blood sample is obtained from the subject.
  • FIG. 4 exemplifies processing and tracking of blood sample components. Each component is tracked with a bar code, dated, is entered into the database and associated with the subject and the profile of the subject. Typically, the whole blood is centrifuged to produce plasma, red blood cells (which pellet) and leukocytes found in the buffy coat which layers in between. Various samples are obtained and coded with a bar code and stored for use as needed.
  • Samples are collected from the subjects.
  • the samples include, but are not limited to, tissues, cells, and fluids, such as nucleic acid, blood, plasma, amniotic fluid, synovial fluid, urine, saliva, aqueous humor, sweat, sperm samples and cerebral spinal fluid. It is understood that the particular set of samples depends upon the organisms in the population.
  • each sample is indexed with an identifier, particularly a machine readable code, such as a bar code.
  • an identifier particularly a machine readable code, such as a bar code.
  • this information is entered into the database in the memory of the storage medium and associated with each subject.
  • This information includes, but is not limited to, genotypic information.
  • nucleic acid sequence information and other information indicative of polymorphisms such as masses of PCR fragments, peptide fragment sequences or masses, spectra of biopolymers and small molecules and other indicia of the structure or function of a gene, gene product or other marker from which the existence of a polymorphism within the population can be inferred.
  • a database can be derived from a collection of blood samples.
  • FIG. 1 shows the status of a collection of over 5000 individual samples. The samples were processed in the laboratory following SOP (standard operating procedure) guidelines. Any standard blood processing protocol can be used.
  • Age At least 17 years old
  • HIV Human immunodeficiency virus
  • a relational database is a an exemplary format in which data are stored as matrices or tables of the parameters linked by an indexer that identifies each subject.
  • Software for preparing and manipulating, including sorting the database can be readily developed or adapted from commercially available software, such as Microsoft Access.
  • Quality control procedures can be implemented. For example, after collection of samples, the quality of the collection in the bank can be assessed. For example, mix-up of samples can be checked by testing for known markers, such as sex. After samples are separated by ethnicity, samples are randomly tested for a marker associated with a particular ethnicity, such as HLA DQA1 group specific component, to assess whether the samples have been properly sorted by ethnic group.
  • An exemplary sample bank is depicted in FIG. 4.
  • Analyzed material include proteins, metabolites, nucleic acids, lipids and any other desired constituent of the material.
  • nucleic acids such as genomic DNA, can be analyzed by sequencing.
  • Sequencing can be performed using any method known to those of skill in the art. For example, if a polymorphism is identified or known, and it is desired to assess its frequency or presence among the subjects in the database, the region of interest from each sample can be isolated, such as by PCR or restriction fragments, hybridization or other suitable method known to those of skill in the art and sequenced.
  • sequencing analysis can be effected using mass spectrometry (see, e.g., U.S. Pat. Nos. 5,547,835, 5,622,824, 5,851,765, and 5,928,906). Nucleic acids also can be sequenced by hybridization (see, e.g., U.S. Pat. Nos. 5,503,980, 5,631,134, 5,795,714) and including analysis by mass spectrometry (see, U.S. application Ser. Nos. 08/419,994 and 09/395,409).
  • Amplification can be performed, e.g., by PCR and/or LCR, according to methods known in the art.
  • genomic DNA of a cell is exposed to two PCR primers and amplification for a number of cycles sufficient to produce the required amount of amplified DNA.
  • the primers are located between 150 and 350 base pairs apart.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • Nucleic acids also can be analyzed by detection methods and protocols, particularly those that rely on mass spectrometry (see, e.g., U.S. Pat. No. 5,605,798, 6,043,031, allowed copending U.S. application Ser. No. 08/744,481, U.S. application Ser. No. 08/990,851 and International PCT application No. WO 99/31278, International PCT application No. WO 98/20019). These methods can be automated (see, e.g., copending U.S. application Ser. No. 09/285,481 and published International PCT application No. PCT/US00/08111, which describes an automated process line).
  • a solid support such as a silicon or silicon-coated substrate, such as in the form of an array
  • analyses are performed using mass spectrometry, particularly MALDI, small nanoliter volumes of sample are loaded on, such that the resulting spot is about, or smaller than, the size of the laser spot. It has been found that when this is achieved, the results from the mass spectrometric analysis are quantitative. The area under the signals in the resulting mass spectra are proportional to concentration (when normalized and corrected for background). Methods for preparing and using such chips are described in U.S. Pat. No. 6,024,925, co-pending U.S. application Ser. Nos.
  • the methods provided herein permit quantitative determination of alleles.
  • the areas under the signals in the mass spectra can be used for quantitative determinations.
  • the frequency is determined from the ratio of the signal to the total area of all of the spectrum and corrected for background. This is possible because of the PROBE technology as described in the above applications incorporated by reference herein.
  • Additional methods of analyzing nucleic acids include amplification-based methods including polymerase chain reaction (PCR), ligase chain reaction (LCR), mini-PCR, rolling circle amplification, autocatalytic methods, such as those using Q ⁇ replicase, TAS, 3SR, and any other suitable method known to those of skill in the art.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • mini-PCR mini-PCR
  • rolling circle amplification such as those using Q ⁇ replicase, TAS, 3SR, and any other suitable method known to those of skill in the art.
  • polymorphisms include but are not limited to, allele specific probes, Southern analyses, and other such analyses.
  • Amplified fragments are yielded by standard polymerase chain methods (U.S. Pat. Nos. 4,683,195 and 4,683,202).
  • the fragmentation method involves the use of enzymes that cleave single or double strands of DNA and enzymes that ligate DNA.
  • the cleavage enzymes can be glycosylases, nickases, and site-specific and non site-specific nucleases, such as, but are not limited to, glycosylases, nickases and site-specific nucleases.
  • DNA glycosylases specifically remove a certain type of nucleobase from a given DNA fragment. These enzymes can thereby produce abasic sites, which can be recognized either by another cleavage enzyme, cleaving the exposed phosphate backbone specifically at the abasic site and producing a set of nucleobase specific fragments indicative of the sequence, or by chemical means, such as alkaline solutions and or heat. The use of one combination of a DNA glycosylase and its targeted nucleotide would be sufficient to generate a base specific signature pattern of any given target region.
  • a DNA glycosylase can be uracil-DNA glycolsylase (UDG) , 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, thymine mismatch-DNA glycosylase, hypoxanthine-DNA glycosylase, 5-Hydroxymethyluracil DNA glycosylase (HmUDG), 5-Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenine DNA glycosylase (see, e.g.,, U.S. Pat. Nos.
  • Uracil for example, can be incorporated into an amplified DNA molecule by amplifying the DNA in the presence of normal DNA precursor nucleotides (e.g. dCTP, dATP, and dGTP) and dUTP.
  • normal DNA precursor nucleotides e.g. dCTP, dATP, and dGTP
  • UDG normal DNA precursor nucleotides
  • uracil residues are cleaved.
  • Subsequent chemical treatment of the products from the UDG reaction results in the cleavage of the phosphate backbone and the generation of nucleobase specific fragments.
  • the separation of the complementary strands of the amplified product prior to glycosylase treatment allows complementary patterns of fragmentation to be generated.
  • dUTP and Uracil DNA glycosylase allows the generation of T specific fragments for the complementary strands, thus providing information on the T as well as the A positions within a given sequence.
  • a C-specific reaction on both (complementary) strands i.e. with a C-specific glycosylase
  • a DNA nickase can be used to recognize and cleave one strand of a DNA duplex.
  • Numerous nickases are known. Among these, for example, are nickase NY2A nickase and NYS1 nickase (Megabase) with the following cleavage sites:
  • NY2A 5′ . . . R AG . . . 3′
  • NYS1 5′ . . . CC[A/G/T] . . . 3′
  • the Fen-ligase method involves two enzymes: Fen-1 enzyme and a ligase.
  • the Fen-1 enzyme is a site-specific nuclease known as a “flap” endonuclease (U.S. Pat. Nos. 5,843,669, 5,874,283, and 6,090,606). This enzyme recognizes and cleaves DNA “flaps” created by the overlap of two oligonucleotides hybridized to a target DNA strand. This cleavage is highly specific and can recognize single base pair mutations, permitting detection of a single homologue from an individual heterozygous at one SNP of interest and then genotyping that homologue at other SNPs occurring within the fragment.
  • Fen-1 enzymes can be Fen-1 like nucleases e.g. human, murine, and Xenopus XPG enzymes and yeast RAD2 nucleases or Fen-1 endonucleases from, for example, M. jannaschii, P. furiosus, and P. woesei. Among such enzymes are the Fen-1 enzymes.
  • the ligase enzyme forms a phosphodiester bond between two double stranded nucleic acid fragments.
  • the ligase can be DNA Ligase I or DNA Ligase IlIl (see, e.g., U.S. Pat. Nos. 5,506,137, 5,700,672, 5,858,705 and 5,976,806; see, also, Waga, et al. (1994) J. Biol. Chem. 269:10923-10934, Li et al. (1994) Nucleic Acids Res. 22:632-638, Arrand et al. (1986) J. Biol. Chem.
  • Restriction enzymes bind specifically to and cleave double-stranded DNA at specific sites within or adjacent to a particular recognition sequence. These enzymes have been classified into three groups (e.g. Types I, II, and III) as known to those of skill in the art. Because of the properties of type I and type III enzymes, they have not been widely used in molecular biological applications. Thus, for purposes herein type II enzymes are among those contemplated. Of the thousands of restriction enzymes known in the art, there are 179 different type II specificities.
  • 31 have a 4-base recognition sequence
  • 11 have a 5-base recognition sequence
  • 127 have a 6-base recognition sequence
  • 10 have recognition sequences of greater than six bases (U.S. Pat. No. 5,604,098).
  • type IIS is exemplified herein.
  • Type IIS enzymes can be Alw XI, Bbv I, Bce 83, Bpm I, Bsg I, Bsm AI, Bsm FI, Bsa I, Bcc I, Bcg I, Ear I, Eco 57I, Esp 3I, Fau I, Fok I, Gsu I, Hga I, Mme I, Mbo II, Sap I, and the otheres.
  • Fok I enzyme endonuclease is an exemplary well characterized member of the Type IIS class (see, e.g., U.S. Pat. Nos. 5,714,330, 5,604,098, 5,436,150, 6,054,276 and 5,871,911; see, also, Szybalski et al. (1991 ) Gene 100:13-26, Wilson and Murray (1991) Ann. Rev. Genet. 25:585-627, Sugisaki et al. (1981) Gene 16:73-78, Podhajska and Szalski (1985) Gene 40:175-182.
  • Fok I recognizes the sequence 5′GGATG-3′ and cleaves DNA accordingly.
  • Type IIS restriction sites can be introduced into DNA targets by incorporating the sites into primers used to amplify such targets. Fragments produced by digestion with Fok I are site specific and can be analyzed by mass spectrometry methods such as MALDI-TOF mass spectrometry, ESI-TOF mass spectrometry, and any other type of mass spectrometry well known to those of skill in the art.
  • age groups can be screened for polymorphisms.
  • allelic dropout is examined by doing comparative PCR in an adjacent region of the genome.
  • allelic frequencies can be determined across the population by analyzing each sample in the population individually, determining the presence or absence of allele or marker of interest in each individual sample, and then determining the frequency of the marker in the population.
  • the database can then be sorted (stratified) to identify any correlations between the allele and a selected parameter using standard statistical analysis. If a correlation is observed, such as a decrease in a particular marker with age or correlation with sex or other parameter, then the marker is a candidate for further study, such as genetic mapping to identify a gene or pathway in which it is involved.
  • the marker can then be correlated, for example, with a disease. Haplotying also can be carried out. Genetic mapping can be effected using standard methods and can also require use of databases of others, such as databases previously determined to be associated with a disorder.
  • the frequency of genotypic and other markers can be obtained by pooling samples.
  • a target population and a genetic variation to be assessed is selected, a plurality of samples of biopolymers are obtained from members of the population, and the biopolymer from which the marker or genotype can be inferred is determined or detected.
  • FIG. 10 depicts the frequency of the CETP Allele in pooled versus individual samples.
  • FIG. 15 shows ethnic diversity among various ethnic groups in the database using pooled DNA samples to obtain the data.
  • FIGS. 12 - 14 show mass spectra for these samples.
  • pooling of test samples has application not only to the healthy databases provided herein, but also to use in gathering data for entry into any database of subjects and genotypic information, including typical databases derived from diseased populations. What is demonstrated herein, is the finding that the results achieved are statistically the same as the results that would be achieved if each sample is analyzed separately. Analysis of pooled samples by a method, such as the mass spectrometric methods provided herein, permits resolution of such data and quantitation of the results.
  • the R53Q acid polymorphism was assessed.
  • the “individual” data represent allelic frequency observed in 92 individuals reactions.
  • the pooled data represent the allelic frequency of the same 92 individuals pooled into a single probe reaction.
  • the concentration of DNA in the samples of individual donors is 250 nanograms.
  • the total concentration of DNA in the pooled samples is also 250 nanograms, where the concentration of any individual DNA is 2.7 nanograms.
  • markers identified thereby can be used, for example, for identification of previously unidentified or unknown genetic markers and to identify new uses for known markers. As markers are identified, these can be entered into the database to use as sorting parameters from which additional correlations can be determined.
  • the samples in the healthy databases can be used to identify new polymorphisms and genetic markers, using any mapping, sequencing, amplification and other methodologies, and in looking for polymorphisms among the population in the database.
  • the thus-identified polymorphism can then be entered into the database for each sample, and the database sorted (stratified) using that polymorphism as a sorting parameter to identify any patterns and correlations that emerge, such as age correlated changes in the frequency of the identified marker. If a correlation is identified, the locus of the marker can be mapped and its function or effect assessed or deduced.
  • the databases can also be used in conjunction with known markers and sorted to identify any correlations.
  • the databases can be used for:
  • diseases and disorders for which polymorphisms can be linked include, those linked to inborn errors of metabolism, acquired metabolic disorders, intermediary metabolism, oncogenesis pathways, blood clotting pathways, and DNA synthetic and repair pathways, DNA repair/replication/transcription factors and activities, e.g., such as genes related to oncogenesis, aging and genes involved in blood clotting and the related biochemical pathways that are related to thrombosis, embolism, stroke, myocardial infarction, angiogenesis and oncogenesis.
  • a number of diseases are caused by or involve deficient or defective enzymes in intermediary metabolism (see, e.q., Tables 1 and 2, below) that result, upon ingestion of the enzyme substrates, in accumulation of harmful metabolites that damage organs and tissues, particularly an infant's developing brain and other organs, resulting in mental retardation and other developmental disorders.
  • the p53 gene is a tumor suppressor gene that is mutated in diverse tumor types.
  • One common allelic variant occurs at codon 72.
  • HDL-C high density lipoprotein cholesterol
  • an exemplary database containing about 5000 subjects, answers to the questionnaire (see FIG. 3), and genotypic information has been stratified.
  • a particular known allele has been selected, and the samples tested for the marker using mass spectrometric analyses, particularly PROBE (see the EXAMPLES) to identify polymorphisms in each sample.
  • the population in the database has been sorted according to various parameters and correlations have been observed. For example, FIGS. 2 A-C, show sorting of the data by age and sex for the Lipoprotein Lipase gene in the Caucasian population in the database. The results show a decrease in the frequency of the allele with age in males but no such decrease in females.
  • Other alleles that have been tested against the database include, alleles of p53, p21 and factor VII. Results when sorted by age are shown in the figures.
  • an age- and sex-stratified population-based sample bank that is ethnically homogenous is a suitable tool for rapid identification and validation of genetic factors regarding their potential medical utility.
  • Systems, including computers, containing the databases are provided herein.
  • the computers and databases can be used in conjunction, for example, with the APL system (see, copending U.S. application Ser. No. 09/285,481), which is an automated system for analyzing biopolymers, particularly nucleic acids. Results from the APL system can be entered into the database.
  • Any suitable computer system can be used.
  • the computer system can be integrated into systems for sample analysis, such as the automated process line described herein (see, e.g., copending U.S. application Ser. No. 09/285,481).
  • FIG. 17 is a block diagram of a computer constructed to provide and process the databases described herein.
  • the processing that maintains the database and performs the methods and procedures can be performed on multiple computers all having a similar construction, or can be performed by a single, integrated computer.
  • the computer through which data are added to the database can be separate from the computer through which the database is sorted, or can be integrated with it. In either arrangement, the computers performing the processing can have a construction as illustrated in FIG. 17.
  • FIG. 17 is a block diagram of an exemplary computer 1700 that maintains the database described above and performs the methods and procedures.
  • Each computer 1700 operates under control of a central processor unit (CPU) 1702, such as a “Pentium” microprocessor and associated integrated circuit chips, available from Intel Corporation of Santa Clara, Calif., USA.
  • CPU central processor unit
  • a computer user can input commands and data from a keyboard and display mouse 1704 and can view inputs and computer output at a display 1706.
  • the display is typically a video monitor or flat panel display device.
  • the computer 1700 also includes a direct access storage device (DASD) 1707, such as a fixed hard disk drive.
  • the memory 1708 typically comprises volatile semiconductor random access memory (RAM).
  • Each computer can include a program product reader 1710 that accepts a program product storage device 1712, from which the program product reader can read data (and to which it can optionally write data).
  • the program product reader can comprise, for example, a disk drive, and the program product storage device can comprise removable storage media such as a magnetic floppy disk, an optical CD-ROM disc, a CD-R disc, a CD-RW disc, or a DVD data disc.
  • the computers can be connected so they can communicate with each other, and with other connected computers, over a network 1713.
  • Each computer 1700 can communicate with the other connected computers over the network 1713 through a network interface 1714 that enables communication over a connection 1716 between the network and the computer.
  • the computer 1700 operates under control of programming steps that are temporarily stored in the memory 1708 in accordance with conventional computer construction.
  • the programming steps can be executed by the CPU 1702, the pertinent system components perform their respective functions.
  • the programming steps implement the functionality of the system as described above.
  • the programming steps can be received from the DASD 1707, through the program product reader 1712, or through the network connection 1716.
  • the storage drive 1710 can receive a program product, read programming steps recorded thereon and transfer the programming steps into the memory 1708 for execution by the CPU 1702.
  • the program product storage device 1710 can comprise any one of multiple removable media having recorded computer-readable instructions, including magnetic floppy disks and CD-ROM storage discs.
  • Other suitable program product storage devices can include magnetic tape and semiconductor memory chips. In this way, the processing steps necessary for operation can be embodied on a program product.
  • the program steps can be received into the operating memory 1708 over the network 1713.
  • the computer receives data including program steps into the memory 1708 through the network interface 1714 after network communication has been established over the network connection 1716 by well-known methods that will be understood by those skilled in the art without further explanation.
  • the program steps are then executed by the CPU 1702 to implement the processing of the Garment Database system.
  • FIG. 18 is a flow diagram that illustrates the processing steps performed using the computer illustrated in FIG. 17, to maintain and provide access to the databases, such as for identifying polymorphic genetic markers.
  • the information contained in the database is stored in computers having a construction similar to that illustrated in FIG. 17.
  • the first step for maintaining the database, as indicated in FIG. 18, is to identify healthy members of a population.
  • the population members are subjects that are selected only on the basis of being healthy, and where the subjects are mammals, such as humans, they can be selected based upon apparent health and the absence of detectable infections.
  • the step of identifying is represented by the flow diagram box numbered 1802.
  • the next step is to obtain identifying and historical information and data relating to the identified members of the population.
  • the information and data comprise parameters for each of the population members, such as member age, ethnicity, sex, medical history, and ultimately genotypic information.
  • the parameter information is obtained from a questionnaire answered by each member, from whom a body tissue or body fluid sample also is obtained.
  • the step of entering and storing these parameters into the database of the computer is represented by the flow diagram box numbered 1806. As additional information about each population member and corresponding sample is obtained, this information can be inputted into the database and can serve as a sorting parameter.
  • the parameters of the members are associated with an indexer.
  • This step can be executed as part of the database storage operation, such as when a new data record is stored according to the relational database structure and is automatically linked with other records according to that structure.
  • the step 1806 also can be executed as part of a conventional data sorting or retrieval process, in which the database entries are searched according to an input search or indexing key value to determine attributes of the data. For example, such search and sort techniques can be used to follow the occurrence of known genetic markers and then determine if there is a correlation with diseases for which they have been implicated. Examples of this use are for assessing the frequencies of the p53 and Lipoprotein Lipase polymorphisms.
  • Such searching of the database also can be valuable for identifying one or more genetic markers whose frequency changes within the population as a function of age, ethnic group, sex, or some other criteria. This can allow the identification of previously unknown polymorphisms and, ultimately, identification of a gene or pathway involved in the onset and progression of disease.
  • the database can be used for taking an identified polymorphism and ascertaining whether it changes in frequency when the data are sorted according to a selected parameter.
  • the databases and methods provided herein permit, among other things, identification of components, particularly key components, of a disease process by understanding its genetic underpinnings, and also an understanding of processes, such as individual drug responses.
  • the databases and methods provided herein also can be used in methods involving elucidation of pathological pathways, in developing new diagnostic assays, identifying new potential drug targets, and in identifying new drug candidates.
  • a database containing information provided by a population of healthy blood donors who were not selected for any particular disease to can be used to identify polymorphisms and the alleles in which they are present, whose frequency decreases with age. These can represent morbidity susceptibility markers and genes.
  • Polymorphisms of the genome can lead to altered gene function, protein function or genome instability. To identify those polymorphisms which have a clinical relevance/utility is the goal of a world-wide scientific effort. It can be expected that the discovery of such polymorphisms will have a fundamental impact on the identification and development of novel drug compounds to cure diseases. The strategy to identify valuable polymorphisms is cumbersome and dependent upon the availability of many large patient and control cohorts to show disease association. In particular, genes that cause a general risk of the population to suffer from any disease (morbidity susceptibility genes) will escape these case/control studies entirely.
  • morbidity susceptibility gene is a gene that is expressed in many different cell types or tissues (housekeeping gene) and its altered function can facilitate the expression of a clinical phenotype caused by disease-specific susceptibility genes that are involved in a pathway specific for this disorder.
  • these morbidity susceptibility genes predispose people to develop a distinct disease according to their genetic make-up for this disease.
  • Candidates for morbidity susceptibility genes can be found at the bottom level of pathways involving transcription, translation, heat-shock proteins, protein trafficking, DNA repair, assembly systems for subcellular structures (e.g. mitochondria, peroxysomes and other cellular microbodies), receptor signaling cascades, immunology, etc. Those pathways control the quality of life at the cellular level as well as for the entire organism. Mutations/polymorphisms located in genes encoding proteins for those pathways can reduce the fitness of cells and make the organism more susceptible to express the clinical phenotype caused by the action of a disease-specific susceptibility gene. Therefore, these morbidity susceptibility genes can be potentially involved in a whole variety of different complex diseases if not in all. Disease-specific susceptibility genes are involved in pathways that can be considered as disease-specific pathways like glucose-, lipid, hormone metabolism, etc.
  • the exemplified method permit, among other things, identification of genes and/or gene products involved in a man's general susceptibility to morbidity and/or mortality; use of these genes and/or gene products in studies to elucidate the genetic underpinnings of human diseases; use of these genes and/or gene products in combinatorial statistical analyses without or together with disease-specific susceptibility genes; use of these genes and/or gene products to predict penetrance of disease susceptibility genes; use of these genes and/or gene products in predisposition and/or acute medical diagnostics and use of these genes and/or gene products to develop drugs to cure diseases and/or to extend the life span of humans.
  • the healthy population stratified by age, gender and ethnicity, etc. is a very efficient and a universal screening tool for morbidity associated genes. Changes of allelic frequencies in the young compared to the old population are expected to indicate putative morbidity susceptibility genes. Individual samples of this healthy population base can be pooled to further increase the throughput. In an experiment, pools of young and old Caucasian females and males were applied to screen more than 400 randomly chosen single nucleotide polymorphisms located in many different genes. Candidate polymorphisms were identified if the allelic difference was greater than 8% between young and old for both or only one of the genders. The initial results were assayed again in at least one independent subsequent experiments.
  • This example describes the use of a database containing information provided by a population of healthy blood donors who were not selected for any particular disease to determine the distribution of allelic frequencies of known genetic markers with age and by sex in a Caucasian subpopulation of the database.
  • the results described in this example demonstrate that a disease-related genetic marker or polymorphism can be identified by sorting a healthy database by a parameter or parameters, such as age, sex and ethnicity.
  • Blood was obtained by venous puncture from human subjects who met blood bank criteria for donating blood.
  • the blood samples were preserved with EDTA at pH 8.0 and labeled.
  • Each donor provided information such as age, sex, ethnicity, medical history and family medical history.
  • Each sample was labeled with a barcode representing identifying information.
  • a database was generated by entering, for each donor, the subject identifier and information corresponding to that subject into the memory of a computer storage medium using commercially available software, e.g., Microsoft Access.
  • N291S polymorphism N291S polymorphism of the Lipoprotein Lipase gene, which results in a substitution of a serine for an asparagine at amino acid codon 291, leads to reduced levels of high density lipoprotein cholesterol (HDL-C) that is associated with an increased risk of males for arteriosclerosis and in particular myocardial infarction (see, Reymer et al. (1995) Nature Genetics 10:28-34).
  • HDL-C high density lipoprotein cholesterol
  • the p53 gene encodes a cell cycle control protein that assesses DNA damage and acts as a transcription factor regulating genes that control cell growth, DNA repair and apoptosis (programmed cell death). Mutations in the p53 gene have been found in a wide variety of different cancers, including different types of leukemia, with varying frequency. The loss of normal p53 function results in genomic instability an uncontrolled cell growth. A polymorphism that has been identified in the p53 gene, i.e., the R72P allele, results in the substitution of a proline for an arginine at amino acid codon 72 of the gene.
  • the p21 gene encodes a cyclin-dependent kinase inhibitor associated with G1phase arrest of normal cells. Expression of the p21 gene triggers apoptosis. Polymorphisms of the p21 gene have been associated with Wilms' tumor, a pediatric kidney cancer. One polymorphism of the p21 gene, the S31R polymorphism, results in a substitution of an arginine for a serine at amino acid codon 31.
  • the genetic polymorphisms were profiled within segments of the Caucasian subpopulation of the sample bank.
  • p53 profiling the genomic DNA isolated from blood from a total of 1277 Caucasian subjects age 18-59 years and 457 Caucasian subjects age 60-79 years was analyzed.
  • p21 profiling the genomic DNA isolated from blood from a total of 910 Caucasian subjects age 18-49 years and 824 Caucasian subjects age 50-79 years was analyzed.
  • lipoprotein lipase gene profiling the genomic DNA from a total of 1464 Caucasian females and 1470 Caucasian males under 60 years of age and a total of 478 Caucasian females and 560 Caucasian males over 60 years of age was analyzed.
  • Genomic DNA was isolated from blood samples obtained from the individuals. Ten milliliters of whole blood from each individual was centrifuged at 2000 ⁇ g. One milliliter of the buffy coat was added to 9 ml of 155 mM NH 4 Cl, 10 mM KHCO 3 , and 0.1 mM Na 2 EDTA, incubated 10 min at room temperature and centrifuged for 10 min at 2000 ⁇ g. The supernatant was removed, and the white cell pellet was washed in 155 mM NH 4 Cl, 10 mM KHCO 3 and 0.1 mM Na 2 EDTA and resuspended in 4.5 ml of 50 mM Tris, 5 mM EDTA and 1% SDS.
  • Proteins were precipitated from the cell lysate by 6 mM ammonium acetate, pH 7.3, and then separated from the nucleic acids by centrifugation at 3000 ⁇ g.
  • the nucleic acid was recovered from the supernatant by the addition of an equal volume of 100% isopropanol and centrifugation at 2000 ⁇ g.
  • the dried nucleic acid pellet was hydrated in 10 mM Tris, pH 7.6, and 1 mM Na 2 EDTA and stored at 4° C.
  • Assays of the genomic DNA to determine the presence or absence of the known genetic markers were developed using the BiomassPROBETM detection method (primer oligo base extension) reaction.
  • This method uses a single detection primer followed by an oligonucleotide extension step to give products, which can be readily resolved by mass spectrometry, and, in particular, MALDI-TOF mass spectrometry.
  • the products differ in length depending on the presence or absence of a polymorphism.
  • a detection primer anneals adjacent to the site of a variable nucleotide or sequence of nucleotides, and the primer is extended using a DNA polymerase in the presence of one or more dideoxyNTPs and, optionally, one or more deoxyNTPs.
  • the resulting products are resolved by MALDI-TOF mass spectrometry.
  • the mass of the products as measured by MALDI-TOF mass spectrometry makes possible the determination of the nucleotide(s) present at the variable site.
  • each of the Caucasian genomic DNA samples was subjected to nucleic acid amplification using primers corresponding to sites 5′ and 3′ of the polymorphic sites of the p21 (S31R allele), p53 (R72P allele) and Lipoprotein Lipase (N291S allele) genes.
  • One primer in each primer pair was biotinylated to permit immobilization of the amplification product to a solid support.
  • the polymerase chain reaction primers used for amplification of the relevant segments of the p21, p53 and lipoprotein lipase genes are shown below: US4p21c31-2F (SEQ ID NO: 9) and US5p21-2R (SEQ ID NO: 10) for p21 gene amplification; US4-p53-ex4-F (also shown as p53-ex4US4 (SEQ ID NO: 2)) and US5-p53/2-4R (also shown as US5P53/4R (SEQ ID NO: 3)) for p53 gene amplification; and US4-LPL-F2 (SEQ ID NO: 16) and US5-LPL-R2 (SEQ ID NO: 17) for lipoprotein lipase gene amplification.
  • Amplification of the respective DNA sequences was conducted according to standard protocols.
  • primers can be used in a concentration of 8 pmol.
  • the reaction mixture e.g., total volume 50 ⁇ l
  • the reaction mixture can contain Taq-polymerase including 10 ⁇ buffer and dTNPs. Cycling conditions for polymerase chain reaction amplification can typically be initially 5 min. at 95° C., followed by 1 min. at 94° C., 45 sec at 53° C., and 30 sec at 72° C. for 40 cycles with a final extension time of 5 min at 72° C.
  • Amplification products can be purified by using Qiagen's PCR purification kit (No. 28106) according to manufacturer's instructions. The elution of the purified products from the column can be done in 50 ⁇ l TE-buffer (10 mM Tris, 1 mM EDTA, pH 7.5).
  • the purified amplification products were immobilized via a biotin-avidin linkage to streptavidin-coated beads and the double-stranded DNA was denatured.
  • a detection primer was then annealed to the immobilized DNA using conditions such as, for example, the following: 50 ⁇ l annealing buffer (20 mM Tris, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 2 , 1% Triton X-100, pH 8) at 50° C. for 10 min, followed by washing of the beads three times with 200 ⁇ l washing buffer (40 mM Tris, 1 mM EDTA, 50 mM NaCl, 0.1% Tween 20, pH 8.8) and once in 200 ⁇ l TE buffer.
  • 50 ⁇ l annealing buffer (20 mM Tris, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 2 , 1% Tri
  • the PROBE extension reaction was performed, for example, by using some components of the DNA sequencing kit from USB (No. 70770) and dNTPs or ddNTPs from Pharmacia.
  • An exemplary protocol could include a total reaction volume of 45 ⁇ l, containing of 21 ⁇ l water, 6 ⁇ l Sequenase-buffer, 3 ⁇ l 10 mM DTT solution, 4.5 ⁇ p, 0.5 mM of three dNTPs, 4.5 ⁇ l, 2 mM the missing one ddNTP, 5.5 ⁇ l glycerol enzyme dilution buffer, 0.25 ⁇ l Sequenase 2.0, and 0.25 pyrophosphatase.
  • the reaction can then by pipetted on ice and incubated for 15 min at room temperature and for 5 min at 37° C.
  • the beads can be washed three times with 200 ⁇ l washing buffer and once with 60 ⁇ l of a 70 mM NH 4 -Citrate solution.
  • the DNA was denatured to release the extended primers from the immobilized template.
  • Each of the resulting extension products was separately analyzed by MALDI-TOF mass spectrometry using 3-hydroxypicolinic acid (3-HPA) as matrix and a UV laser.
  • the primers used in the PROBE reactions are as shown below: P21/31-3 (SEQ ID NO: 12) for PROBE analysis of the p21 polymorphic site; P53/72 (SEQ ID NO: 4) for PROBE analysis of the p53 polymorphic site; and LPL-2 for PROBE analysis of the lipoprotein lipase gene polymorphic site.
  • P21/31-3 SEQ ID NO: 12
  • P53/72 SEQ ID NO: 4
  • LPL-2 for PROBE analysis of the lipoprotein lipase gene polymorphic site.
  • the extension reaction was performed using dideoxy-C.
  • the products resulting from the reaction conducted on a “wild-type” allele template (wherein codon 31 encodes a serine) and from the reaction conducted on a polymorphic S31R allele template (wherein codon 31 encodes an arginine) are shown below and designated as P21/31-3 Ser (wt) (SEQ ID NO: 13) and P21/31-3 Arg (SEQ ID NO: 14), respectively.
  • the masses for each product as can be measured by MALDI-TOF mass spectrometry are also provided (i.e., 4900.2 Da for the wild-type product and 5213.4 Da for the polymorphic product).
  • the extension reaction was performed using a mixture of ddA and ddT.
  • the products resulting from the reaction conducted on a “wild-type” allele template (wherein codon 291 encodes an asparagine) and from the reaction conducted on a polymorphic N291S allele template (wherein codon 291 encodes a serine) are shown below and designated as 291Asn and 291Ser, respectively.
  • the masses for each product as can be measured by MALDI-TOF mass spectrometry are also provided (i.e., 6438.2 Da for the wild-type product and 6758.4 Da for the polymorphic product).
  • Biotinylated US5 primer is used in the PCR amplification.
  • Masses Allele Product Termination ddA, ddT SEQ # Length Mass LPL-2 caatctgggctatgagatca 19 20 6141 291 Asn caatctgggctatgagatcaa 20 21 6438.2 291 Ser caatctgggctatgagatcagt 21 22 6758.4
  • Biotinylated US5 primer is used in the PCR amplification.
  • P21-1 (S31R) Amino acid exchange serine to arginine at codon 31 of the tumor suppressor gene p21.
  • Product length 207 bp US4p21c3l-2F gtcc gtcagaaccc atgcggcagc (SEQ ID NO: 8) p21/31-3 31S aaggcctgcc gccgctctt cggcccagtg ga cagcgagc agctgag ccg cgactgtgat a 31R gcgctaatgg cgggctgcat ccaggaggcc cgtgagcgat ggaacttcga ctttgtcacc gagacaccac tggaggg US5p21-2R Primers (SEQ ID NOs: 9-11) US4p21c31-2F
  • Masses Allele Product Termination ddC SEQ # Length Mass P21/31-3 cagcgagcagctgag 12 15 4627 P21/31-3 Ser cagcgagcagctgagc 13 16 4900.2 (wt) P21/31-3 Arg cagcgagcagctgagac 14 17 5213.4
  • Biotinylated US5 primer is used in the PCR amplification.
  • Each of the Caucasian subject DNA samples was individually analyzed by MALDI-TOF mass spectrometry to determine the identity of the nucleotide at the polymorphic sites.
  • the genotypic results of each assay can be entered into the database.
  • the results were then sorted according to age and/or sex to determine the distribution of allelic frequencies by age and/or sex. As depicted in the Figures showing histograms of the results, in each case, there was a differential distribution of the allelic frequencies of the genetic markers for the p21, p53 and lipoprotein lipase gene polymorphisms.
  • FIG. 8 shows the results of the p21 genetic marker assays and reveals a statistically significant decrease (from 13.3% to 9.2%) in the frequency of the heterozygous genotype (S31 R) in Caucasians with age (18-49 years of age compared to 50-79 years of age).
  • the frequencies of the homozygous (S31 and R31) genotypes for the two age groups are also shown, as are the overall frequencies of the S31 and R31 alleles in the two age groups (designated as *S31 and *R31, respectively in the Figure).
  • FIGS. 7 A-C show the results of the p53 genetic marker assays and reveals a statistically significant decrease (from 6.7% to 3.7%) in the frequency of the homozygous polymorphic genotype (P72) in Caucasians with age (18-59 years of age compared to 60-79 years of age).
  • the frequencies of the homozygous “wild-type” genotype (R72) and the heterozygous genotype (R72P) for the two age groups are also shown, as are the overall frequencies of the R72 and P72 alleles in the two age groups (designated as *R72 and *P72, respectively in the Figure).
  • FIG. 2C shows the results of the lipoprotein lipase gene genetic marker assays and reveals a statistically significant decrease (from 1.97% to 0.54%) in the frequency of the polymorphic allele (S291) in Caucasian males with age (see also Reymer et al. (1995) Nature Genetics 10:28-34). The frequencies of this allele in Caucasian females of different age groups are also shown.
  • This example describes the use of MALDI-TOF mass spectrometry to analyze DNA samples of a number of subjects as individual samples and as pooled samples of multiple subjects to assess the presence or absence of a polymorphic allele (the 353Q allele) of the Factor VII gene and determine the frequency of the allele in the group of subjects.
  • the results of this study show that essentially the same allelic frequency can be obtained by analyzing pooled DNA samples as by analyzing each sample separately and thereby demonstrate the quantitative nature of MALDI-TOF mass spectrometry in the analysis of nucleic acids.
  • Factor VII is a serine protease involved in the extrinsic blood coagulation cascade. This factor is activated by thrombin and works with tissue factor (Factor III) in the processing of Factor X to Factor Xa.
  • tissue factor Factor III
  • the polymorphism investigated in this study is R353Q (i.e., a substitution of a glutamic acid residue for an arginine residue at codon 353 of the Factor VII gene) (see Table 5).
  • Genomic DNA was isolated from separate blood samples obtained from a large number of subjects divided into multiple groups of 92 subjects per group. Each sample of genomic DNA was analyzed using the BiomassPROBETM assay as described in Example 1 to determine the presence or absence of the 353Q polymorphism of the Factor VII gene.
  • DNA from each sample was amplified in a polymerase chain reaction using primers F7-353FUS4 (SEQ ID NO: 24) and F7-353RUS5 (SEQ ID NO: 26) as shown below and using standard conditions, for example, as described in Example 1.
  • One of the primers was biotinylated to permit immobilization of the amplification product to a solid support.
  • the purified amplification products were immobilized via a biotin-avidin linkage to streptavidin-coated beads and the double-stranded DNA was denatured.
  • a detection primer was then annealed to the immobilized DNA using conditions such as, for example, described in Example 1.
  • the detection primer is shown as F7-353-P (SEQ ID NO: 27) below.
  • the PROBE extension reaction was carried out using conditions, for example, such as those described in Example 1. The reaction was performed using ddG.
  • the DNA was denatured to release the extended primers from the immobilized template.
  • Each of the resulting extension products was separately analyzed by MALDI-TOF mass spectrometry.
  • a matrix such as 3-hydroxypicolinic acid (3-HPA) and a UV laser could be used in the MALDI-TOF mass spectrometric analysis.
  • the products resulting from the reaction conducted on a “wild-type” allele template (wherein codon 353 encodes an arginine) and from the reaction conducted on a polymorphic 353Q allele template (wherein codon 353 encodes a glutamic acid) are shown below and designated as 353 CGG and 353 CAG, respectively.
  • the masses for each product as can be measured by MALDI-TOF mass spectrometry are also provided (i.e., 5646.8 Da for the wild-type product and 5960 Da for the polymorphic product).
  • the samples from 92 subjects were pooled (250 nanograms total concentration of DNA in which the concentration of any individual DNA is 2.7 nanograms), and the pool of DNA was subjected to MALDI-TOF mass spectrometric analysis.
  • the area under the signal corresponding to the mass of the 353Q polymorphism PROBE extension product in the resulting spectrum was integrated in order to quantitate the amount of DNA present.
  • the ratio of this amount to total DNA was used to determine the allelic frequency of the 353Q polymorphism in the group of subjects.
  • This type of individual sample vs. pooled sample analysis was repeated for numerous different groups of 92 different samples.
  • Masses Allele Product Termination ddG SEQ # Length Mass F7-353-P atgccacccactacc 27 18 5333.6 353 CGG cacatgccacccactaccg 28 19 5646.8 353 CAG cacatgccacccactaccag 29 20 5960 US5-bio bio- agcggataacaatttcacagg 30 23 7648.6
  • Blood is obtained from a donor by venous puncture and preserved with 1 mM EDTA pH 8.0. Ten milliliters of whole blood from each donor was centrifuged at 2000 ⁇ g. One milliliter of the buffy coat was added to 9 milliters of 155 mM NH 4 Cl, 1 OmM KHCO 3 , and 0.1 mM Na 2 EDTA, incubated 10 minutes at room temperature and centrifuged for 10 minutes at 2000 ⁇ g.
  • the supernatant was removed, and the white cell pellet was washed in 155 mM NH 4 Cl, 10 mM KHCO 3 , and 0.1 mM Na 2 EDTA and resuspended in 4.5 milliliters of 50 mM Tris, 5 mM EDTA, and 1% SDS. Proteins were precipitated from the cell lysate by 6M Ammonium Acetate, pH 7.3, and separated from the nucleic acid by centrifugation 3000 ⁇ g. The nucleic acid was recovered from the supernatant by the addition of an equal volume of 100% isopropanol and centrifugation at 2000 ⁇ g. The dried nucleic acid pellet was hydrated in lOmM Tris pH 7.6 and 1 mM Na2EDTA and stored at 4C.
  • Candidate morbidity and mortality markers include housekeeping genes, such as genes involved in signal transduction.
  • genes include the A-kinase anchoring proteins (AKAPs) genes, which participate in signal transduction pathways involving protein phosphorylation.
  • AKAPs A-kinase anchoring proteins
  • Protein phosphorylation is an important mechanism for enzyme regulation and the transduction of extracellular signals across the cell membrane in eukaryotic cells.
  • a wide variety of cellular substrates, including enzymes, membrane receptors, ion channels and transcription factors, can be phosphorylated in response to extracellular signals that interact with cells.
  • a key enzyme in the phosphorylation of cellular proteins in response to hormones and neurotransmitters is cyclic AMP (cAMP)-dependent protein kinase (PKA).
  • cAMP cyclic AMP
  • PKA cyclic AMP
  • PKA Upon activation by cAMP, PKA thus mediates a variety of cellular responses to such extracellular signals.
  • An array of PKA isozymes are expressed in mammalian cells.
  • the PKAs usually exist as inactive tetramers containing a regulatory (R) subunit dimer and two catalytic (C) subunits.
  • R regulatory
  • C catalytic
  • Genes encoding three C subunits (C ⁇ , C ⁇ and Cy) and four R subunits (RI ⁇ , RI ⁇ , RII ⁇ and RII ⁇ ) have been identified [see Takio et al. (1982) Proc. Natl. Acad. Sci. U.S. A. 79:2544-2548; Lee et al. (1983) Proc. Natl. Acad. Sci.
  • the type I PKA holoenzyme (RI ⁇ and RI ⁇ ) is predominantly cytoplasmic, whereas the majority of type II PKA (RII ⁇ and RII ⁇ ) associates with cellular structures and organelles [Scott (1991) Pharmacol. Ther. 50:123-1451. Many hormones and other signals act through receptors to generate cAMP which binds to the R subunits of PKA and releases and activates the C subunits to phosphorylate proteins. Because protein kinases and their substrates are widely distributed throughout cells, there are mechanisms in place in cells to localize protein kinase-mediated responses to different signals.
  • AKAPs A-kinase anchoring proteins
  • Anchoring not only places the kinase close to the substrates, but also positions the PKA holoenzyme at sites where it can optimally respond to fluctuations in the second messenger cAMP [Mochly-Rosen (1995) Science 268:247-251; Faux and Scott (1996) Trends Biochem. Sci. 21:312-315; Hubbard and Cohen (1993) Trends Biochem. Sci. 18:172-177].
  • RII regulatory subunit
  • AKAPs Up to 75% of type II PKA is localized to various intracellular sites through association of the regulatory subunit (RII) with AKAPs [see, e.g., Hausken et al. (1996) J. Biol. Chem. 271:29016-290221.
  • RII subunits of PKA bind to AKAPs with nanomolar affinity [Carr et al. (1992) J. Biol. Chem. 267:13376-13382], and many AKAP-RII complexes have been isolated from cell extracts.
  • RI subunits of PKA bind to AKAPs with only micromolar affinity [Burton et al. (1997) Proc. Natl. Acad. Sci. U.S.A.
  • AKAPs More than 20 AKAPs have been reported in different tissues and species.
  • Complementary DNAs (cDNAs) encoding AKAPs have been isolated from diverse species, ranging from Caenorhabditis elegans and Drosophilia to human [see, e.g., Colledge and Scott (1999) Trends Cell Biol. 9:216-2211. Regions within AKAPs that mediate association with RII subunits of PKA have been identified. These regions of approximately 10-18 amino acid residues vary substantially in primary sequence, but secondary structure predictions indicate that they are likely to form an amphipathic helix with hydrophobic residues aligned along one face of the helix and charged residues along the other [Carr et al. (1991) J.
  • Hydrophobic amino acids with a long aliphatic side chain e.g., valine, leucine or isoleucine, can participate in binding to RII subunits [Glantz et al. (1993) J. Biol. Chem. 268:12796-12804].
  • AKAPs also have the ability to bind to multiple proteins, including other signaling enzymes.
  • AKAP79 binds to PKA, protein kinase C (PKC) and the protein phosphatase calcineurin (PP2B) [Coghlan et al. (1995) Science 267:108-112 and Klauck et al. (1996) Science 271:1589-15921. Therefore, the targeting of AKAP79 to neuronal postsynaptic membranes brings together enzymes with opposite catalytic activities in a single complex.
  • PKA protein kinase C
  • P2B protein phosphatase calcineurin
  • AKAPs thus serve as potential regulatory mechanisms that increase the selectivity and intensity of a cAMP-mediated response. There is a need, therefore, to identify and elucidate the structural and functional properties of AKAPs in order to gain a complete understanding of the important role these proteins ⁇ play in the basic functioning of cells.
  • AKAP10 cDNA also referred to as D-AKAP2
  • GenBank database accession numbers AF037439 (SEQ ID NO: 31) and NM 007202.
  • the AKAP10 gene is located on chromosome 17.
  • mouse D-AKAP2 cDNA The sequence of a mouse D-AKAP2 cDNA is also available in the GenBank database (see accession number AF021833).
  • the mouse D-AKAP2 protein contains an RGS domain near the amino terminus that is characteristic of proteins that interact with G ⁇ subunits and possess GTPase activating protein-like activity [Huang et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:11184-11189].
  • the human AKAP10 protein also has sequences homologous to RGS domains. The carboxy-terminal 40 residues of the mouse D-AKAP2 protein are responsible for the interaction with the regulatory subunits of PKA. This sequence is fairly well conserved between the mouse D-AKAP2 and human AKAP10 proteins.
  • Polymorphisms of AKAP genes that alter gene expression, regulation, protein structure and/or protein function are more likely to have a significant effect on the regulation of enzyme (particularly PKA) activity, cellular transduction of signals and responses thereto and on the basic functioning of cells than polymorphisms that do not alter gene and/or protein function. Included in the polymorphic AKAPs provided herein are human AKAP10 proteins containing differing amino acid residues at position number 646.
  • Amino acid 646 of the human AKAP10 protein is located in the carboxy-terminal region of the protein within a segment that participates in the binding of R-subunits of PKAs. This segment includes the carboxy-terminal 40 amino acids.
  • the amino acid residue reported for position 646 of the human AKAP10 protein is an isoleucine.
  • Polymorphic human AKAP10 proteins provided herein have the amino acid sequence but contain residues other than isoleucine at amino acid position 646 of the protein.
  • the amino acid at position 646 is a valine, leucine or phenylalanine residue.
  • an allele of the human AKAP10 gene that contains a specific polymorphism at position 2073 of the coding sequence and thereby encodes a valine at position 646 has been detected in varying frequencies in DNA samples from younger and older segments of the human population.
  • the A at position 2073 of the AKAP10 gene coding sequence is changed from an A to a G, giving rise to an altered sequence in which the codon for amino acid 646 changes from ATT, coding for isoleucine, to GTT, coding for valine.
  • Morbidity Marker 1 Human Protein Kinase A Anchoring Protein (AKAP10-1)
  • PCR primers were synthesized by OPERON using phosphoramidite chemistry. Amplification of the AKAP10 target sequence was carried out in single 50 ⁇ l PCR reaction with 100 ng-1 ug of pooled human genomic DNAs in a 50 ⁇ l PCR reaction. Individual DNA concentrations within the pooled samples were present in equal concentration with the final concentration ranging from 1-25 ng.
  • the 5′ biotinylated universal primer After an initial round of amplification with the target with the specific forward and reverse primer, the 5′ biotinylated universal primer then hybridized and acted as a reverse primer thereby introducing a 3′ biotin capture moiety into the molecule.
  • the amplification protocol results in a 5′-biotinylated double stranded DNA amplicon and dramatically reduces the cost of high throughput genotyping by eliminating the need to 5′ biotin label each forward primer used in a genotyping.
  • Thermal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (calculated temperature) with the following cycling parameters: 94° C. for 5 min; 45 cycles: 94° C. for 20 sec, 56° C. for 30 sec, 72° C. for 60 sec; 72° C. 3 min.
  • Genotyping using the BiomassPROBE assay methods was carried out by resuspending the DNA coated magnetic beads in 26 mM Tris-HCl pH 9.5, 6.5 mM MgCl 2 and 50 mM each of dTTP and 50 mM each of ddCTP, ddATP, ddGTP, 2.5U of a thermostable DNA polymerase (Ambersham) and 20 pmol of a template specific oligonucleotide PROBE primer 5′-CTGGCGCCCACGTGGTCAA-3′ (SEQ ID NO: 48) (Operon). Primer extension occurs with three cycles of oligonucleotide primer hybridization and extension.
  • the extension products were analyzed after denaturation from the template with 50 mM NH 4 Cl and transfer of 150 nL each sample to a silicon chip preloaded with 150 nL of H3PA matrix material.
  • the sample material was allowed to crystallize and was analyzed by MALDI-TOF (Bruker, PerSeptive).
  • the SNP that is present in AKAP10-1 is a T to C transversion at nucleotide number 156277 of the sequence of a genomic clone of the AKAP10 gene (GenBank Accession No. AC005730) (SEQ ID NO: 36).
  • SEQ ID NO: 35 represents the nucleotide sequence of human chromosome 17, which contains the genomic nucleotide sequence of the human AKAP10 gene
  • SEQ ID NO: 36 represents the nucleotide sequence of human chromosome 17, which contains the genomic nucleotide sequence of the human AKAP10-1 allele.
  • the mass of the primer used in the BioMass probe reaction was 5500.6 daltons.
  • the primer is extended by the addition of ddC, which has a mass of 5773.8.
  • the wildtype gene results in the addition of dT and ddG to the primer to produce an extension product having a mass of 6101 daltons.
  • the polymorphism is localized in the non-translated 3′-region of the gene encoding the human protein kinase A anchoring protein (AKAP10).
  • the gene is located on chromosome 17. Its structure includes 15 exons and 14 intervening sequences (introns).
  • the encoded protein is responsible for the sub-cellular localization of the cAMP-dependent protein kinase and, therefore, plays a key role in the G-protein mediated receptor-signaling pathway (Huang et al. PNAS (1007) 94:11184-11189).
  • Morbitity Marker 2 Human Protein Kinase A Anchoring Protein (AKAP10-5)
  • Genomic DNA was isolated from blood (as described above) of seventeen (17) individuals with a genotype CC at the AKAP10-1 gene locus and a single heterozygous individual (CT) (as described).
  • a target sequence in the AKAP10-1 gene which encodes the C-terminal PKA binding domain was amplified using the polymerase chain reaction.
  • PCR primers were synthesized by OPERON using phosphoramidite chemistry. Amplification of the AKAP10-1 target sequence was carried out in individual 50 ⁇ l PCR reaction with 25 ng of human genomic DNA templates.
  • Thermal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (MJ Research, Waltham, Mass.) (calculated temperature) with the following cycling parameters: 94° C. for 5 min; 45 cycles; 94° C. for 20 sec, 56° C. for 30 sec, 72° C. for 60 sec; 72° C. 3 min. After amplification the amplicons were purified using a chromatography (Mo Bio Laboratories (Solana Beach, Calif.)).
  • the sequence of the 18 amplicons, representing the target region was determined using a standard Sanger cycle sequencing method with 25 nmol of the PCR amplicon, 3.2 uM DNA sequencing primer 5′-CCC ACA GCA GTT AAT CCT TC-3′(SEQ ID NO: 55), and chain terminating dRhodamine labeled 2′, 3′ dideoxynucleotides (PE Biosystems, Foster City, Calif.) using the following cycling parameters: 96° C. for 15 seconds; 25 cycles: 55° C. for 15 seconds, 60° C. for 4 minutes.
  • the sequencing products precipitated by 0.3M NaOAc and ethanol. The precipitate was centrifuged and dried. The pellets were resuspended in deionized formamide and separated on a 5% polyacrylimide gel. The sequence was determined using the “Sequencher” software (Gene Codes, Ann Arbor, Mich.).
  • AKAP10-5 SEQ ID NO: 33
  • SEQ ID NO: 33 This single nucleotide polymorphism was designated as AKAP10-5 (SEQ ID NO: 33) and resulted in a substitution of a valine for an isoleucine residue at amino acid position 646 of the amino acid sequence of human AKAP10 (SEQ ID NO: 32).
  • the healthy population stratified by age is a very efficient and a universal screening tool for morbidity associated genes by allowing for the detection of changes of allelic frequencies in the young compared to the old population. Individual samples of this healthy population base can be pooled to further increase the throughput.
  • Healthy samples were obtained through the blood bank of San Bernardino, Calif. Both parents of the blood donors were of Caucasian origin. Practically a healthy subject, when human, is defined as human donor who passes blood bank criteria to donate blood for eventual use in the general population. These criteria are as follows: free of detectable viral, bacterial, mycoplasma, and parasitic infections; not anemic; and then further selected based upon a questionnaire regarding history (see FIG. 3). Thus, a healthy population represents an unbiased population of sufficient health to donate blood according to blood bank criteria, and not further selected for any disease state. Typically such individuals are not taking any medications.
  • PCR primers were synthesized by OPERON using phosphoramidite chemistry. Amplification of the AKAP10 target sequence was carried out in a single 50 ⁇ l PCR reaction with 100 ng-1 ⁇ g of pooled human genomic DNAs in a 50 ⁇ l PCR reaction. Individual DNA concentrations within the pooled samples were present in equal concentration with the final concentration ranging from 1-25 ng.
  • Each reaction contained 1 ⁇ PCR buffer (Qiagen, Valencia, Calif.), 200 ⁇ M dNTPs, 1U Hotstar Taq polymerase (Qiagen, Valencia, Calif.), 4 mM MgCl 2 , and 25 pmol of the forward primer containing the universal primer sequence and the target specific sequence 5′-AGCGGATAACAATTTCACACAGGGAGCTAGCTTGGAAGAT TGC-3′ (SEQ ID NO: 41), 2 pmol of the reverse primer 5′-GTCCAATATATGCAAACAGTTG-3′ (SEQ ID NO: 54), and 10 pmol of a biotinylated universal primer complementary to the 5′ end of the PCR amplicon BIO:5′-AGCGGATAACAATTTCACACAGG-3′ (SEQ ID NO: 43).
  • the 5′ biotinylated universal primer can then be hybridized and acted as a forward primer thereby introducing a 5′ biotin capture moiety into the molecule.
  • the amplification protocol resulted in a 5′-biotinylated double stranded DNA amplicon and dramatically reduced the cost of high throughput genotyping by eliminating the need to 5′ biotin label every forward primer used in a genotyping.
  • Themal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (calculated temperature) with the following cycling parameters: 94° C. for 5 min; 45 cycles: 94° C. for 20 sec, 56° C. for 30 sec; 72° C. for 60 sec; 72° C. 3 min.
  • BiomassPROBETM assay of primer extension analysis (see, U.S. Pat. No. 6,043,031) of donor population for AKAP 10-5 (SEQ ID NO: 33) was performed. Genotyping using these methods was carried out by resuspending the DNA coated magnetic beads in 26 mM Tris-HCL pH 9.5, 6.5 mM MgCl 2 , 50 mM dTTP, 50 mM each of ddCTP, ddATP, ddGTP, 2.5U of a thermostable DNA polymerase (Ambersham), and 20 pmol of a template specific oligonucleotide PROBE primer 5′-ACTGAGCCTGCTGCATAA-3′ (SEQ ID NO: 44) (Operon).
  • Primer extension occurs with three cycles of oligonucleotide primer with hybridization and extension.
  • the extension products were analyzed after denaturation from the template with 50 mM NH 4 Cl and transfer of 150 nL of each sample to a silicon chip preloaded with 150 nl of H3PA matrix material.
  • the sample material was allowed to crystallize and analyzed by MALDI-TOF (Bruker, PerSeptive).
  • the primer has a mass of 5483.6 daltons.
  • the SNP results in the addition of a ddC to the primer, giving a mass of 5756.8 daltons for the extended product.
  • the wild type results in the addition a T and ddG to the primer giving a mass of 6101 daltons.
  • the frequency of the SNP was measured in a population of age selected healthy individuals. Seven hundred thirteen (713) individuals under 40 years of age (360 females, 353 males) and 703 individuals over 60 years of age (322 females, 381 males) were tested for the presence of the SNP, AKAP10-5 (SEQ ID NO: 33). Results are presented below in Table 4.
  • FIG. 20 graphically shows these results of allele and genotype distribution in the age and sex stratified Caucasian population.
  • Morbidity Marker 3 Human Methionine Sulfoxide Reductase A (msrA)
  • PCR primers were synthesized by OPERON using phosphoramidite chemistry. Amplification of the AKAP10 target sequence was carried out in single 50 ⁇ l PCR reaction with 100 ng-1 ug of pooled human genomic DNA templates in a 50 ⁇ l PCR reaction. Individual DNA concentrations within the pooled samples were present in an equal concentration with the final concentration ranging from 1-25 ng.
  • the 5′ biotinylated universal primer was then hybridized and acted as a reverse primer thereby introducing a 3′ biotin capture moiety into the molecule.
  • the amplification protocol results in a 5′-biotinylated double stranded DNA amplicon and dramatically reduces the cost of high throughput genotyping by eliminating the need to 5′ biotin label each forward primer used in a genotyping.
  • Thermal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (calculated temperature) with the following cycling parameters: 94° C. for 5 min; 45 cycles: 94° C. for 20 sec, 56° C. for 30 sec, 72° C. for 60 sec; 72° C. 3 min.
  • Genotyping using the BiomassPROBE assay methods was carried out by resuspending the DNA coated magnetic beads in 26 mM Tris-HCl pH 9.5, 6.5 mM MgCl 2 , 50 mM of dTTPs and 50 mM each of ddCTP, ddATP, ddGTP, 2.5U of a thermostable DNA polymerase (Ambersham), and 20 pmol of a template specific oligonucleotide PROBE primer 5′-CTGAAAAGGGAGAGAAAG-3′ (Operon) (SEQ ID NO: 52). Primer extension occurs with three cycles of oligonucleotide primer with hybridization and extension.
  • the extension products were analyzed after denaturation from the template with 50 mM NH 4 Cl and transfer of 150 nl each sample to a silicon chip preloaded with 150 nl of H3PA matrix material.
  • the sample material was allowed to crystallize and analyzed by MALDI-TOF (Bruker, PerSeptive).
  • the SNP is represented as a T to C tranversion in the sequence of two ESTs.
  • the wild type is represented by having a T at position 128 of GenBank Accession No. AW 195104, which represents the nucleotide sequence of an EST which is a portion of the wild type human msrA gene (SEQ ID NO: 39).
  • the SNP is presented as a C at position 129 of GenBank Accession No. AW 874187, which represents the nucleotide sequence of an EST which is a portion of an allele of the human msrA gene (SEQ ID NO: 40).
  • the SNP is represented as an A to G transversion.
  • the primer utilized in the BioMass probe reaction had a mass of 5654.8 daltons.
  • the primer is extended by the incorporation of a ddC and has a mass of 5928.
  • the primer is extended by adding a dT and a DDC to produce a mass of 6232.1 daltons.
  • the frequency of the SNP was measured in a population of age selected healthy individuals. Five hundred fifty-two (552) individuals between the ages of 18-39 years (276 females, 276 males and 552 individuals between the age of 60-79 (184 females between the ages of 60-69, 368 males between the age of 60-79) were tested for the presence of the polymorphism localized in the nontranslated 3′ region of h-msr-A.
  • Genotype difference between male age group among healthy individuals is significant.
  • the age-related allele and genotype frequency of this marker in both genders and the entire population is shown in FIG. 21.
  • the decrease of the homozygous CC genotype in the older male population is highly significant.
  • the polymorphism is localized in the non-translated 3′-region of the gene encoding the human methionine sulfoxide reductase (h-msrA). The exact localization is 451 base pairs downstream the stop codon (TAA). It is likely that this SNP is in linkage disequilibrium (LD) with another polymorphism more upstream in the coding or promoter region; thus, it does not directly cause morbidity.
  • the enzyme methionine sulfoxide reductase has been proposed to exhibit multiple biological functions. It can serve to repair oxidative protein damage but also play an important role in the regulation of proteins by activation or inactivation of their biological functions (Moskovitz et al.
  • the products of the enzymatic digestions were purified with ZipTips (Millipore, Bedford, Mass.).
  • the ZipTips were pre-wetted with 10 ⁇ L 50% acetonitrile and equilibrated 4 times with 10 ⁇ l 0.1 M TEAAc.
  • the oligonucleotide fragments were bound to the C18 in the ZipTip material by continuous aspiration and dispension of each sample into the ZipTip.
  • Each digested oligonucleotide was conditioned by washing with 10 ⁇ L 0.1 M TEAAc, followed by 4 washing steps with 10 ⁇ L H 2 O. DNA fragments were eluted from the Ziptip with 7 ⁇ L 50% acetonitrile.
  • any method for condition the samples can be employed.
  • Methods for conditioning which generally is used to increase peak resolution, are well known (see, e.g., International PCT application No. WO 98/20019).
  • DNA Glycosylases modifies DNA at each position that a specific nucleobase resides in the DNA, thereby producing abasic sites. In a subsequent reaction with another enzyme, a chemical, or heat, the phosphate backbone at each abasic site can be cleaved.
  • the glycosylase utilized in the following procedures was uracil-DNA glycosylase (UDG).
  • Uracil bases were incorporated into DNA fragments in each position that a thymine base would normally occupy by amplifying a DNA target sequence in the presence of uracil.
  • Each uracil substituted DNA amplicon was incubated with UDG, which cleaved each uracil base in the amplicon, and was then subjected to conditions that effected backbone cleavage at each abasic site, which produced DNA fragments.
  • DNA fragments were subjected to MALDI-TOF mass spectrometry analysis. Genetic variability in the target DNA was then assessed by analyzing mass spectra.
  • Glycosylases specific for nucleotide analogs or modified nucleotides, as described herein, can be substituted for UDG in the following procedures.
  • the glycosylase methods described hereafter, in conjunction with phosphate backbone cleavage and MALDI, can be used to analyze DNA fragments for the purposes of SNP scanning, bacteria typing, methylation analysis, microsatellite analysis, genotyping, and nucleotide sequencing and re-sequencing.
  • a glycosylase procedure was used to genotype the DNA sequence encoding UCP-2 (Uncoupling Protein 2).
  • the sequence for UCP-2 is deposited in GenBank under accession number AF096289.
  • the sequence variation genotyped in the following procedure was a cytosine (C-allele) to thymine (T-allele) variation at nucleotide position 4790, which results in a alanine to valine mutation at position 55 in the UCP-2 polypeptide.
  • DNA was amplified using a PCR procedure with a 50 ⁇ L reaction volume containing of 5 pmol biotinylated primer having the sequence 5′-TGCTTATCCCTGTAGCTACCCTGTCTTGGCCTTGCAGATCCAA-3′ (SEQ ID NO: 91), 15 pmol non-biotinylated primer having the sequence 5′-AGCGGATAACAATTTCACACAGGCCATCACACCGCGGTACTG-3′ (SEQ ID NO: 92), 200 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 600 ⁇ M dUTP (to fully replace dTTP), 1.5 mM to 3 mM MgCl 2 , 1 U of HotStarTaq polymerase, and 25 ng of CEPH DNA. Amplification was effected with 45 cycles at an annealing temperature of 56° C.
  • the amplification product was then immobilized onto a solid support by incubating 50 ⁇ L of the amplification reaction with 5 ⁇ L of prewashed Dynabeads for 20 minutes at room temperature. The supernatant was removed, and the beads were incubated with 50 ⁇ L of 0.1 M NaOH for 5 minutes at room temperature to denature the double-stranded PCR product in such a fashion that single-stranded DNA was linked to the beads. The beads were then neutralized by three washes with 50 ⁇ L 10 mM TrisHCl (pH 8).
  • the beads were resuspended in 10 ⁇ L of a 60 mM TrisHCl/1 mM EDTA (pH 7.9) solution, and 1 U uracil DNA glycosylase was added to the solution for 45 minutes at 37° C. to remove uracil nucleotides present in the single-stranded DNA linked to the beads.
  • the beads were then washed two times with 25 ⁇ L of 10 mM TrisHCl (pH 8) and once with 10 ⁇ L of water.
  • the biotinylated strands were then eluted from the beads with 12 ⁇ L of 2 M NH 4 OH at 60° C. for 10 minutes.
  • the backbone of the DNA was cleaved by incubating the samples for 10 min at 95° C. (with a closed lid), and ammonia was evaporated from the samples by incubating the samples for 11 min at 80° C.
  • the glycosylase assay was conducted using pooled samples to detect genetic variability at the UCP-2 locus. DNA of known genotype was pooled from eleven individuals and was diluted to a fixed concentration of 5 ng/ ⁇ L. The procedure provided in Example 3A was followed using 2 pmol of forward primer having a sequence of 5′-CCCAGTCACGACGTTGTAAAACGTCTTGGCCTTGCAGATCCAAG-3′ (SEQ ID NO: 93) and 15 pmol of reverse primer having the sequence 5′-AGCGGATAACAATTTCACACAGGCCATCACACCGCGGTACTG-3′ (SEQ ID NO: 94).
  • biotinylated primer having the sequence 5′bioCCCAGTCACGACGTTGTAAAACG 3′ can be introduced to the PCR reaction after about two cycles.
  • the fragments were analyzed via MALDI-TOF mass spectroscopy (Example 4).
  • the T-allele which generated a unique fragment of 3254 Daltons, could be distinguished in mass spectra from the C-allele, which generated a unique fragment of 4788 Daltons.
  • Allelic frequency in the pooled samples was quantified by integrating the area under each signal corresponding to an allelic fragment. Integration was accomplished by hand calculations using equations well known to those skilled in the art. In the pool of eleven samples, this procedure suggested that 40.9% of the individuals harbored the T allele and 59.09% of the individuals harbored the C allele.
  • BKR-2 Bradykinin Receptor 2
  • the sequence for BKR-2 is deposited in GenBank under accession number X86173.
  • BKR-2 includes a SNP in the promoter region, which is a C to T variation, as well as a SNP in a repeated unit, which is a G to T variation.
  • the procedure provided in Example 3A was utilized to identify the SNP in the promotor region, the SNP in the microsattelite repeat region, and the number of repeated units in the microsattelite region of BKR-2.
  • a forward PCR primer having the sequence 5′-CTCCAGCTGGGCAGGAGTGC-3′ (SEQ ID NO: 95) and a reverse primer having the sequence 5′-CACTTCAGTCGCTCCCT-3′ (SEQ ID NO: 96) were utilized to amplify BKR-2 DNA in the presence of uracil.
  • the amplicon was fragmented by UDG followed by backbone cleavage.
  • the cleavage fragments were analyzed by MALDI-TOF mass spectrometry as described in Example 4.
  • the T-allele generated a unique fragment having a mass of 1784 Daltons, which was readily detected in a mass spectrum. Hence, the presence of the T-allele was indicative of the G to T sequence variation in the repeat region of BKR-2.
  • the number of repeat regions was distinguished between individuals having two repeat sequences and individuals having three repeat sequences in BKR-2.
  • the DNA of these individuals did not harbor the G to T sequence variation in the repeat sequence as each repeat sequence contained a G at the SNP locus.
  • the number of repeat regions was determined in individual samples by calculating the area under a signal corresponding to a unique DNA fragment having a mass of 2771.6 Daltons. This signal in spectra generated from individuals having two repeat regions had an area that was thirty-three percent less than the area under the same signal in spectra generated from individuals having three repeat regions.
  • the procedures discussed above can be utilized to genotype individuals for the number of repeat sequences present in BKR-2.
  • Bisulfite treatment of genomic DNA can be utilized to analyze positions of methylated cytosine residues within the DNA. Treating nucleic acids with bisulfite deaminates cytosine residues to uracil residues, while methylated cytosine remains unmodified. Thus, by comparing the sequence of a PCR product generated from genomic DNA that is not treated with bisulfite with the sequence of a PCR product generated from genomic DNA that is treated with bisulfite, the degree of methylation in a nucleic acid as well as the positions where cytosine is methylated can be deduced.
  • Genomic DNA (2 ⁇ g) was digested by incubation with 1 ⁇ L of a restriction enzyme at 37° C. for 2 hours. An aliquot of 3 M NaOH was added to yield a final concentration of 0.3M NaOH in the digestion solution. The reaction was incubated at 37° C. for 15 minutes followed by treatment with 5.35M urea, 4.44M bisulfite, and 10 mM hydroquinone, where the final concentration of hydroquinone is 0.5 mM.
  • sample A The sample that was treated with bisulfite was compared to the same digestion sample that had not undergone bisulfite treatment (sample B).
  • sample A and sample B were amplified by a standard PCR procedure.
  • the PCR procedure included the step of overlaying each sample with mineral oil and then subjecting the sample to thermocycling (20 cycles of 15 minutes at 55° C. followed by 30 seconds at 95° C.).
  • the PCR reaction contained four nucleotide bases, C, A, G, and U.
  • the mineral oil was removed from each sample, and the PCR products were purified with glassmilk. Sodium iodide (3 volumes) and glassmilk (5 ⁇ L) were added to samples A and B.
  • the samples were then placed on ice for 8 minutes, washed with 420 ⁇ L cold buffer, centrifuged for 10 seconds, and the supernatant fractions were removed. This process was repeated twice and then 25 ⁇ L of water was added. Samples were incubated for 5 minutes at 37° C., were centrifuged for 20 seconds, and the supernatant fraction was collected, and then this incubation/centrifugation/supernatant fraction collection procedure was repeated. 50 ⁇ L 0.1 M NaOH was then added to the samples to denature the DNA.
  • sample A and sample B were then treated with 2U of UDG (MBI Fermentas) and then subjected to backbone cleavage, as described herein.
  • UDG MBI Fermentas
  • the resulting fragments from each of sample A and sample B were analyzed by MALDI-TOF mass spectroscopy as described in Example 4.
  • Sample A gave rise to a greater number of fragments than the number of fragments arising from sample B, indicative that the nucleic acid harbored at least one methylated cytosine moiety.
  • Haplotyping procedures permit the selection of a fragment from one of an individual's two homologous chromosomes and to genotype linked SNPs on that fragment.
  • the direct resolution of haplotypes can yield increased information content, improving the diagnosis of any linked disease genes or identifying linkages associated with those diseases.
  • haplotypes were typically reconstructed indirectly through pedigree analysis (in cases where pedigrees were available) through laborious and unreliable allele-specific PCR or through single-molecule dilution methods well known in the art.
  • a haplotyping procedure was used to determine the presence of two SNPs, referred to as SNP1 and SNP2, located on one strand in a DNA sample.
  • the haplotyping procedure used in this assay utilized Fen-1, a site-specific “flap” endonuclease that cleaves DNA “flaps” created by the overlap of two oligonucleotides hybridized to a target DNA strand.
  • the two overlapping oligonucleotides in this example were short arm and long arm allele-specific adaptors.
  • the target DNA was an amplified nucleic acid that had been denatured and contained SNP1 and SNP2.
  • the short arm adaptor included a unique sequence not found in the target DNA.
  • the 3′ distal nucleotide of the short arm adaptor was identical to one of the SNP1 alleles.
  • the long arm adaptor included two regions: a 3′ region complementary to the short arm and a 5′ gene-specific region complementary to the fragment of interest adjacent to the SNP. If there was a match between the adaptor and one of the homologues, the Fen enzyme recognized and cleaved the overlapping flap.
  • the short arm of the adaptor was then ligated to the remainder of the target fragment (minus the SNP site). This ligated fragment was used as the forward primer for a second PCR reaction in which only the ligated homologue was amplified.
  • the second PCR product (PCR2) was then analyzed by mass spectrometry. If there was no match between the adaptors and the target DNA, there was no overlap, no cleavage by Fen-1, and thus no PCR2 product of interest.
  • the second SNP was found by using an adaptor that was specific for SNP2 and hybridizing the adaptor to the PCR2 product containing the first SNP.
  • the Fen-ligase and amplification procedures were repeated for the PCR2 product containing the first SNP. If the amplified product yielded a second SNP, then SNP1 and SNP2 were on the same fragment.
  • the SNP is unknown, then four allele-specific adaptors (e.g. C, G, A, and T) can be used to hybridize with the target DNA.
  • the substrates are then treated with the Fen-ligase protocol, including amplification.
  • the PCR2 products can be analyzed by PROBE, as described herein, to determine which adaptors were hybridized to the DNA target and thus identify the SNPs in the sequence.
  • a Fen-ligase assay was used to detect two SNPs present in Factor VII. These SNPs are located 814 base pairs apart from each other. SNP1 was located at position 8401 (C to T), and SNP2 was located at 9215 (G to A).
  • a PCR product (PCR1) was generated for a known heterozygous individual at SNP1, a short distance from the 5′ end of the SNP. Specifically, a 10 ⁇ L PCR reaction was performed by mixing 1.5 mM MgCl 2 , 200 ⁇ M of each dNTP, 0.5 U HotStar polymerase, 0.1 ⁇ M of a forward primer having the sequence 5′-GCG CTC CTG TCG GTG CCA (SEQ ID NO: 56), 0.1 ⁇ M of a reverse primer having the sequence 5′-GCC TGA CTG GTG GGG CCC (SEQ ID NO: 57), and 1 ng of genomic DNA. The annealing temperature was 58° C., and the amplification process yielded fragments that were 861 bp in length.
  • the PCR1 reaction mixture was divided in half and was treated with an exonuclease 1/SAP mixture (0.22 ⁇ L mixture/5 ⁇ L PCR1 reaction) which contained 1.0 ⁇ L SAP and 0.1 ⁇ L exon1.
  • the exonuclease treatment was done for 30 minutes at 37° C. and then 20 minutes at 85° C. to denature the DNA.
  • C and T allele-specific adaptors
  • the long arm and short arm oligonucleotides of each adaptor (10 ⁇ M) were mixed in a 1:1 ratio and heated for 30 seconds at 95° C. The temperature was reduced in 2° C. increments to 37° C. for annealing.
  • the C-adaptor had a short arm sequence of 5′-CAT GCA TGC ACG GTC (SEQ ID NO: 58) and a long arm sequence of 5′-CAG AGA GTA CCC CTC GAC CGT GCA TGC ATG (SEQ ID NO: 59).
  • the long arm of the adaptor was 30 bp (15 bp gene-specific), and the short arm was 15 bp.
  • the T-adaptor had a short arm sequence of 5′-CAT GCA TGC ACG GTT (SEQ ID NO: 60) and a long arm sequence of 5′-GTA CGT ACG TGC CAA CTC CCC ATG AGA GAC (SEQ ID NO: 61).
  • the adaptor could also have a hairpin structure in which the short and long arm are separated by a loop containing of 3 to 10 nucleotides (SEQ ID NO: 118).
  • Solution B A second solution (Solution B) containing of 1.65 ⁇ l Ampligase (Thermostable ligase, Epicentre Technologies), 1.65 ⁇ l 200 ng/ ⁇ l MFEN (from Methanocuccus jannaschil), and 3.0 ⁇ l of an allel specific adaptor (C or T) was prepared.
  • Solution B was added to Solution A at 95° C. and incubated at 55° C. for 3 hours.
  • the total reaction volume was 15.0 ⁇ l per adaptor-specific reaction. For a bi-allelic system, 2 ⁇ 15.0 ⁇ l reactions were required.
  • a second amplification reaction was conducted in each sample tube using the short arm adaptor (C or T) sequence as the forward primer (minus the SNP1 site). Only the ligated homologue was amplified.
  • a standard PCR reaction was conducted with a total volume of 10.0 ⁇ l containing of 1 ⁇ Buffer (final concentration), 1.5 mM final concentration MgCl 2 , 200 ⁇ M final concentration dNTPs, 0.5 U HotStar polymerase, 0.1 ⁇ M final concentration forward primer 5′-CAT GCA TGC ACG GT (SEQ ID NO: 62), 0.1 ⁇ M final concentration reverse primer 5′-GCC TGA CTG GTG GGG CCC (SEQ ID NO: 63), and 1.0 ⁇ l of the purified FEN-ligase reaction solution. The annealing temperature was 58° C.
  • the PCR2 product was analyzed by MALDI TOF mass spectroscopy as described in Example 4. The mass spectrum of Fen SNP1 showed a mass of 6084.08 Daltons
  • the second SNP (SNP2) can be found by using an adaptor that is specific for SNP2 and hybridizing that adaptor to the PCR2 product containing the first SNP.
  • the Fen-ligase and amplification procedures are repeated for the PCR2 product containing the first SNP. If the amplified product yields a second SNP, then SN1 and SN2 are on the same fragment.
  • the mass spectrum of SNP2, representing the T allele, showed a mass of 6359.88 Daltons.
  • This assay also can be performed upon pooled DNA to yield haplotype frequencies as described herein.
  • the Fen-ligase assay can be used to analyze multiplexes as described herein.
  • NY2A nickase and NYS1 nickase (Megabase), which cleave DNA at the following sites:
  • NY2A 5′ . . . R AG . . . 3′
  • NYS1 5′ . . . ⁇ CC[A/G/T] . . . 3′
  • Tris-HCl (10 mM), KCl (10 mM, pH 8.3), magnesium acetate (25 mM), BSA (1 mg/mL), and 6 U of Cvi NY2A or Cvi NYS1 Nickase (Megabase Research) were added to 25 pmol of double-stranded oligonucleotide template having a sequence of 5′-CGC AGG GTT TCC TCG TCG CAC TGG GCA TGT G-3′ (SEQ ID NO: 90, Operon, Alameda, Calif.) synthesized using standard phosphoramidite chemistry. With a total volume of 20 ⁇ L, the reaction mixture was incubated at 37° C.
  • Example 5 The digestion products were purified using ZipTips (Millipore, Bedford, Mass.) as described in Example 5.
  • the samples were analyzed by MALDI-TOF mass spectroscopy as described in Example 1.
  • the nickase Cvi NY2A yielded three fragments with masses 4049.76 Daltons, 5473.14 Daltons, and 9540.71 Daltons.
  • the Cvi NYS1 nickase yielded fragments with masses 2063.18 Daltons, 3056.48 Daltons, 6492.81 Daltons, and 7450.14 Daltons.
  • DQA was amplified from the genomic DNA of 100 healthy individuals.
  • DQA was amplified using standard PCR chemistry in a reaction having a total volume of 50 ⁇ L containing of 10 mM Tris-HCl, 10 mM KCl (pH 8.3), 2.5 mM MgCl 2 , 200 ⁇ M of each dNTP, 10 pmol of a forward primer having the sequence 5′-GTG CTG CAG GTG TAA ACT TGT ACC AG-3′(SEQ ID NO: 64), 10 pmol of a reverse primer having the sequence 5′-CAC GGA TCC GGT AGC AGC GGT AGA GTT G-3′(SEQ ID NO: 65), 1 U DNA polymerase (Stoffel fragment, Perkin Elmer), and 200 ng human genomic DNA (2 ng DNA/individual).
  • the template was denatured at 94° C. for 5 minutes. Thermal cycling was continued with a touch-down program that included 45 cycles of 20 seconds at 94° C., 30 seconds at 56° C., 1 minute at 72° C., and a final extension of 3 minutes at 72° C.
  • the crude PCR product was used in the subsequent nickase reaction.
  • the unpurified PCR product was subjected to nickase digestion. Tris-HCl (10 mM), KCl (10 mM, pH 8.3), magnesium acetate (25 mM), BSA (1 mg/mL), and 5 U of Cvi NY2A or Cvi NYS1 Nickase (Megabase Research) were added to 25 pmol of the amplified template with a total reaction volume of 20 ⁇ L. The mixture was then incubated at 37° C. for 5 hours. The digestion products were purified with either ZipTips (Millipore, Bedford, Mass.) as described in Example 5. The samples were analyzed by MALDI-TOF mass spectroscopy as described in Example 4. This assay also can be used to do multiplexing and standardless genotyping as described herein.
  • the two complementary strands can be separated after digestion by using a single-stranded undigested PCR product as a capture probe.
  • This probe (preparation shown below in Example 8C) can be hybridized to the nickase fragments in hybridization buffer containing 200 mM sodium citrate and 1% blocking reagent (Boehringer Mannheim). The reaction is heated to 95° C. for 5 minutes and cooled to room temperature over 30 minutes by using a thermal cycler (PTC-200 DNA engine, MJ Research, Waltham, Mass.). The capture probe-nickase fragment is immobilized on 140 ⁇ g of streptavidin-coated magnetic beads.
  • the beads are subsequently washed three times with 70 mM ammonium citrate.
  • the captured single-stranded nickase fragments are eluted by heating to 80° C. for 5 minutes in 5 ⁇ L of 50 mM ammonium hydroxide.
  • the capture probe is prepared by amplifying the human ⁇ -globin gene (3′ end of intron 1 to 5′ end of exon 2) via PCR methods in a total volume of 50 ⁇ L containing of GeneAmp 1XPCR Buffer II, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl 2 , 0.2 mM dNTP mix, 10 pmol of each primer (forward primer 5′-ACTGGGCATGTGGAGACAG-3′(SEQ ID NO: 66) and biotinylated reverse primer bio5′-GCACTTTCTTGCCATGAG-3′(SEQ ID: 67), 2 U of AmpliTaq Gold, and 200 ng of human genomic DNA.
  • GeneAmp 1XPCR Buffer II 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl 2 , 0.2 mM dNTP mix, 10 pmol of each primer (forward primer 5′-ACT
  • the template is denatured at 94° C. for 8 minutes. Thermal cycling is continued with a touch-down program that included 11 cycles of 20 seconds at 94° C., 30 seconds at 64° C., 1 minute at 72° C.; and a final extension of 5 minutes at 72° C.
  • the amplicon is purified using UltraCleanTM PCR clean-up kit (MO Bio Laboratories, Solano Beach, Calif.).
  • a Type IIS assay was used to identify human gene sequences with known SNPs.
  • the Type IIS enzyme used in this assay was Fok I which effected double-stranded cleavage of the target DNA.
  • the assay involved the steps of amplification and Fok I treatment of the amplicon.
  • the primers were designed so that each PCR product of a designated gene target was less than 100 bases such that a Fok I recognition sequence was incorporated at the 5′ and 3′ end of the amplicon. Therefore, the fragments that were cleaved by Fok I included a center fragment containing the SNP of interest.
  • Amplification of the ten human gene sequences were carried out in a single 50 ⁇ L volume PCR reaction with 20 ng of human genomic DNA template in 5 PCR reaction tubes.
  • Each reaction vial contained 1 ⁇ PCR buffer (Qiagen), 200 ⁇ M dNTPs, 1 U Hotstar Taq polymerase (Qiagen), 4 mM MgCl 2 , and 10 pmol of each primer.
  • the primers were designed such that a Fok I recognition site was incorporated at the 5′ and 3′ ends of the amplicon.
  • Thermal cycling was performed in 0.2 mL tubes or a 96 well plate using a MJ Research Thermal Cycler (calculated temperature) with the following cycling parameters: 94° C. for 5 minutes; 45 cycles: 94° C. for 20 seconds, 56° C. for 20 seconds, 72° C. for 60 seconds; and 72° C. for 3 minutes.
  • the sample was treated with 0.2 U Exonuclease I (Amersham Pharmacia) and S Alkaline Phosphotase (Amersham Pharmacia) to remove the unincorporated primers and dNTPs.
  • 0.2 U of exonuclease I and SAP were added to 5 ⁇ L of the PCR sample. The sample was then incubated at 37° C. for 15 minutes. Exonuclease I and SAP were then inactivated by heating the sample up to 85° C. for 15 minutes.
  • Fok I digestion was performed by adding 2 U of Fok I (New England Biolab) to the 5 uL PCR sample and incubating at 37° C. for 30 minutes.
  • a healthy database can be used to associate a disease state with a specific allele (SNP) that has been found to show a strong association between age and the allele, in particular the homozygous genotype.
  • SNP specific allele
  • the method involves using the same healthy database used to identify the age dependent association, however stratification is by information given by the donors about common disorders from which their parents suffered (the donor's familial history of disease). There are three possible answers a donor could give about the health status of their parents: neither were affected, one was affected or both were affected. Only donors above a certain minimum age, depending on the disease, are utilized, as the donors parents must be old enough to to have exhibited clinical disease phenotypes. The genotype frequency in each of these groups is determined and compared with each other. If there is an association of the marker in the donor to a disease the frequency of the heterozyous genotype will be increased. The frequency of the homozygous genotype should not increase, as it should be significantly underrepresented in the healthy population.
  • the apparatus 10 for identifying a biological sample generally comprises a mass spectrometer 15 communicating with a computing device 20.
  • the mass spectrometer can be a MALDI-TOF mass spectrometer manufactured by Bruker-Franzen Analytik GmbH; however, it will be appreciated that other mass spectrometers can be substituted.
  • the computing device 20 is typically a general purpose computing device. It will be appreciated that the computing device could be alternatively configured, for example, it can be integrated with the mass spectrometer or could be part of a computer in a larger network system.
  • the apparatus 10 for identifying a biological sample can operate as an automated identification system having a robot 25 with a robotic arm 27 configured to deliver a sample plate 29 into a receiving area 31 of the mass spectrometer 15.
  • the sample to be identified can be placed on the plate 29 and automatically received into the mass spectrometer 15.
  • the biological sample is then processed in the mass spectrometer to generate data indicative of the mass of DNA fragments in the biological sample.
  • This data can be sent directly to computing device 20, or can have some preprocessing or filtering performed within the mass spectrometer.
  • the mass spectrometer 15 transmits unprocessed and unfiltered mass spectrometry data to the computing device 20. It will be appreciated that the analysis in the computing device can be adjusted to accommodate preprocessing or filtering performed within the mass spectrometer.
  • method 35 data are received into a computing device from a test instrument in block 40.
  • data are received in a raw, unprocessed and unfiltered form, but alternatively can have some form of filtering or processing applied.
  • the test instrument of an exemplary embodiment is a mass spectrometer as described above. It will be appreciated that other test instruments could be substituted for the mass spectrometer.
  • the data generated by the test instrument, and in particular the mass spectrometer includes information indicative of the identification of the biological sample. More specifically, the data are indicative of the DNA composition of the biological sample.
  • mass spectrometry data gathered from DNA samples obtained from DNA amplification techniques are noisier than, for example, those from typical protein samples. This is due in part because protein samples are more readily prepared in more abundance, and protein samples are more easily ionizable as compared to DNA samples. Accordingly, conventional mass spectrometer data analysis techniques are generally ineffective for DNA analysis of a biological sample. To improve the analysis capability so that DNA composition data can be more readily discerned, an embodiment uses wavelet technology for analyzing the DNA mass spectrometry data.
  • Wavelets are an analytical tool for signal processing, numerical analysis, and mathematical modeling. Wavelet technology provides a basic expansion function which is applied to a data set. Using wavelet decomposition, the data set can be simultaneously analyzed in the time and frequency domains. Wavelet transformation is the technique of choice in the analysis of data that exhibit complicated time (mass) and frequency domain information, such as MALDI-TOF DNA data. Wavelet transforms as described herein have superior denoising properties as compared to conventional Fourier analysis techniques. Wavelet transformation has proven to be particularly effective in interpreting the inherently noisy MALDI-TOF spectra of DNA samples. In using wavelets, a “small wave” or “scaling function” is used to transform a data set into stages, with each stage representing a frequency component in the data set. Using wavelet transformation, mass spectrometry data can be processed, filtered, and analyzed with sufficient discrimination to be useful for identification of the DNA composition for a biological sample.
  • the data received in block 40 is denoised in block 45.
  • the denoised data then has a baseline correction applied in block 50.
  • a baseline correction is generally necessary as data coming from the test instrument, in particular a mass spectrometer instrument, has data arranged in a generally exponentially decaying manner. This generally exponential decaying arrangement is not due to the composition of the biological sample, but is a result of the physical properties and characteristics of the test instrument, and other chemicals involved in DNA sample preparation. Accordingly, baseline correction substantially corrects the data to remove a component of the data attributable to the test system, and sample preparation characteristics.
  • a signal remains which is generally indicative of the composition of the biological sample. Due to the extraordinary discrimination required for analyzing the DNA composition of the biological sample, the composition is not readily apparent from the denoised and corrected signal. For example, although the signal can include peak areas, it is not yet clear whether these “putative” peaks actually represent a DNA composition, or whether the putative peaks are the result of a systemic or chemical aberration. Further, any call of the composition of the biological sample would have a probability of error which would be unacceptable for clinical or therapeutic purposes. In such critical situations, there needs to be a high degree of certainty that any call or identification of the sample is accurate. Therefore, additional data processing and interpretation is necessary before the sample can be accurately and confidently identified.
  • the biological sample is selected and processed to have only a limited range of possible compositions. Accordingly, it is therefore known where peaks indicating composition should be located, if present. Taking advantage of knowing the location of these expected peaks, in block 60 the method 35 matches putative peaks in the processed signal to the location of the expected peaks. In such a manner, the probability of each putative peak in the data being an actual peak indicative of the composition of the biological sample can be determined. Once the probability of each peak is determined in block 60, then in block 65 the method 35 statistically determines the composition of the biological sample, and determines if confidence is high enough to calling a genotype.
  • FIG. 26 shows an example of data from a mass spectrometer.
  • the mass spectrometer data 70 generally comprises data points distributed along an x-axis 71 and a y-axis 72.
  • the x-axis 71 represents the mass of particles detected, while the y-axis 72 represents a numerical concentration of the particles.
  • the mass spectrometry data 70 is generally exponentially decaying with data at the left end of the x-axis 73 generally decaying in an exponential manner toward data at the heavier end 74 of the x-axis 71.
  • the general exponential presentation of the data is not indicative of the composition of the biological sample, but is more reflective of systematic error and characteristics. Further, as described above and illustrated in FIG. 26, considerable noise exists in the mass spectrometry DNA data 70.
  • the denoising process generally entails 1) performing a wavelet transformation on the raw data to decompose the raw data into wavelet stage coefficients; 2) generating a noise profile from the highest stage of wavelet coefficients; and 3) applying a scaled noise profile to other stages in the wavelet transformation.
  • the denoising process is further described below.
  • the wavelet transformation of the raw mass spectrometry data is generally diagramed.
  • the mass spectrometry data 70 is sequentially transformed into stages. In each stage, the data are represented in a high stage and a low stage, with the low stage acting as the input to the next sequential stage.
  • the mass spectrometry data 70 is transformed into stage 0 high data 82 and stage 0 low data 83.
  • the stage 0 low data 83 is then used as an input to the next level transformation to generate stage 1 high data 84 and stage 1 low data 85.
  • the stage 1 low data 85 is used as an input to be transformed into stage 2 high data 86 and stage 2 low data 87.
  • the transformation is continued until no more useful information can be derived by further wavelet transformation.
  • a 24-point wavelet is used. More particularly a wavelet commonly referred to as the Daubechies 24 is used to decompose the raw data. It will be appreciated that other wavelets can be used for the wavelet transformation. Since each stage in a wavelet transformation has one-half the data points of the previous stage, the wavelet transformation can be continued until the stage n low data 89 has around 50 points. Accordingly, the stage n high 88 would contain about 100 data points. Since the exemplary wavelet is 24 points long, little data or information can be derived by continuing the wavelet transformation on a data set of around 50 points.
  • FIG. 28 shows an example of stage 0 high data 95. Since stage 0 high data 95 is generally indicative of the highest frequencies in the mass spectrometry data, stage 0 high data 95 will closely relate to the quantity of high frequency noise in the mass spectrometry data.
  • an exponential fitting formula has been applied to the stage 0 high data 95 to generate a stage 0 noise profile 97.
  • the exponential fitting formula is in the format A 0 +A 1 EXP ( ⁇ A 2 m). It will be appreciated that other exponential fitting formulae or other types of curve fits can be used.
  • noise profiles for the other high stages are determined. Since the later data points in each stage will likely be representative of the level of noise in each stage, only the later data points in each stage are used to generate a standard deviation figure that is representative of the noise content in that particular stage. More particularly, in generating the noise profile for each remaining stage, only the last five percent of the data points in each stage are analyzed to determined a standard deviation number. It will be appreciated that other numbers of points, or alternative methods could be used to generate such a standard deviation figure.
  • stage 0 noise profile (the exponential curve) 97 to generate a scaled noise profile for each stage.
  • FIG. 30 shows that stage 1 high data 98 has stage 1 high data 103 with the last five percent of the data points represented by area 99. The points in area 99 are evaluated to determine a standard deviation number indicative of the noise content in stage 1 high data 103. The standard deviation number is then used with the stage 0 noise profile 97 to generate a stage 1 noise profile.
  • stage 2 high 100 has stage 2 high data 104 with the last five percent of points represented by area 101.
  • the data points in area 101 are then used to calculate a standard deviation number which is then used to scale the stage 0 noise profile 97 to generate a noise profile for stage 2 data.
  • stage n high data 108 has the last five percent of data points indicated in area 106.
  • the data points in area 106 are used to determine a standard deviation number for stage n.
  • the stage n standard deviation number is then used with the stage 0 noise profile 97 to generate a noise profile for stage n. Accordingly, each of the high data stages has a noise profile.
  • FIG. 31 shows how the noise profile is applied to the data in each stage.
  • the noise profile is used to generate a threshold which is applied to the data in each stage. Since the noise profile is already scaled to adjust for the noise content of each stage, calculating a threshold permits further adjustment to tune the quantity of noise removed. Wavelet coefficients below the threshold are ignored while those above the threshold are retained. Accordingly, the remaining data have a substantial portion of the noise content removed.
  • stage 0 and 1 Due to the characteristics of wavelet transformation, the lower stages, such as stage 0 and 1, will have more noise content than the later stages such as stage 2 or stage n. Indeed, stage n low data are likely to have little noise at all. Therefore, in an embodiment, the noise profiles are applied more aggressively in the lower stages and less aggressively in the later stages.
  • FIG. 31 shows that stage 0 high threshold is determined by multiplying the stage 0 noise profile by a factor of four. In such a manner, significant numbers of data points in stage 0 high data 95 will be below the threshold and therefore eliminated.
  • Stage 1 high threshold 112 is set at two times the noise profile for the stage 1 high data, and stage 2 high threshold 114 is set equal to the noise profile for stage 2 high.
  • stage n high threshold 116 is therefore determined by scaling the noise profile for each respective stage n high by a factor equal to (1 ⁇ 2 n-2 ). It will be appreciated that other factors can be applied to scale the noise profile for each stage. For example, the noise profile can be scaled more or less aggressively to accommodate specific systemic characteristics or sample compositions. As indicated above, stage n low data does not have a noise profile applied as stage n low data 118 is assumed to have little or no noise content. After the scaled noise profiles have been applied to each high data stage, the mass spectrometry data 70 has been denoised and is ready for further processing. A wavelet transformation of the denoised signal results in the sparse data set 120 as shown in FIG. 31.
  • the mass spectrometry data received in block 40 has been denoised in block 45 and is now passed to block 50 for baseline correction.
  • the artifacts introduced by the wavelet transformation procedure can be removed.
  • Wavelet transformation results vary slightly depending upon which point of the wavelet is used as a starting point.
  • an exemplary embodiment uses the 24-point Daubechies-24 wavelet.
  • the denoised data are transformed using every available possible starting point, with the results averaged to determine a final denoised and shifted signal.
  • FIG. 33 shows that the wavelet coefficient is applied 24 different times and then the results averaged to generate the final data set. It will be appreciated that other techniques can be used to accommodate the slight error introduced due to wavelet shifting.
  • FIG. 33 shows an example of the wavelet coefficient 135 data set from the denoised and shifted signal 130.
  • FIG. 36 shows that putative peak areas 145, 147, and 149 are located in the denoised and shifted signal 150.
  • the putative peak areas are systematically identified by taking a moving average along the signal 150 and identifying sections of the signal 150 which exceed a threshold related to the moving average. It will be appreciated that other methods can be used to identify putative peak areas in the signal 150.
  • Putative peak areas 145, 147 and 149 are removed from the signal 150 to create a peak-free signal 155 as shown in FIG. 37.
  • the peak-free signal 155 is further analyzed to identify remaining minimum values 157, and the remaining minimum values 157 are connected to generate the peak-free signal 155.
  • FIG. 38 shows a process of using the peak-free signal 155 to generate a baseline 170 as shown in FIG. 39.
  • a wavelet transformation is performed on the peak-free signal 155. All the stages from the wavelet transformation are eliminated in block 164 except for the n low stage.
  • the n low stage will generally indicate the lowest frequency component of the peak-free signal 155 and therefore will generally indicate the system exponential characteristics.
  • Block 166 shows that a signal is reconstructed from the n low coefficients and the baseline signal 170 is generated in block 168.
  • FIG. 39 shows a denoised and shifted data signal 172 positioned adjacent a correction baseline 170.
  • the baseline correction 170 is subtracted from the denoised and shifted signal 172 to generate a signal 175 having a baseline correction applied as shown in FIG. 40.
  • the putative peaks in signal 175 are not identifiable with sufficient accuracy or confidence to call the DNA composition of a biological sample.
  • the data from the baseline correction 50 is now compressed in block 55; the compression technique used in an exemplary embodiment is detailed in FIG. 41.
  • the data in the baseline corrected data are presented in an array format 182 with x-axis points 183 having an associated data value 184.
  • the x-axis is indexed by the non-zero wavelet coefficients, and the associated value is the value of the wavelet coefficient.
  • the maximum value 184 is indicated to be 1000.
  • an intermediate format 186 is generated.
  • the intermediate format 186 generally comprises a real number having a whole number portion 188 and a decimal portion 190.
  • the whole number portion is the x-axis point 183 while the decimal portion is the value data 184 divided by the maximum data value.
  • a data value “25” is indicated at x-axis point “100” .
  • the intermediate value for this data point would be “100.025”.
  • the final compressed data 195 is generated.
  • the first point of the intermediate data file becomes the starting point for the compressed data.
  • each data point in the compressed data 195 is calculated as follows: the whole number portion (left of the decimal) is replaced by the difference between the current and the last whole number. The remainder (right of the decimal) remains intact.
  • the starting point of the compressed data 195 is shown to be the same as the intermediate data point which is “100.025”.
  • the comparison between the first intermediate data point “100.025” and the second intermediate data point “150.220” is “50.220”. Therefore, “50.220” becomes the second point of the compressed data 195.
  • the second intermediate point is “150.220” and the third intermediate data point is “500.0001” . Therefore, the third compressed data becomes “350.000”.
  • the calculation for determining compressed data points is continued until the entire array of data points is converted to a single array of real numbers.
  • FIG. 42 generally describes the method of compressing mass spectrometry data, showing that the data file in block 201 is presented as an array of coefficients in block 202. The data starting point and maximum is determined as shown in block 203, and the intermediate real numbers are calculated in block 204 as described above. With the intermediate data points generated, the compressed data are generated in block 205.
  • the described compression method is highly advantageous and efficient for compressing data sets such as a processed data set from a mass spectrometry instrument.
  • the method is particularly useful for data, such as mass spectrometry data, that uses large numbers and has been processed to have occasional lengthy gaps in x-axis data. Accordingly, an x-y data array for processed mass spectrometry data can be stored with an effective compression rate of 10 ⁇ or more.
  • the compression technique is applied to mass spectrometry data, it will be appreciated that the method can also advantageously be applied to other data sets.
  • peak heights are now determined in block 60.
  • the first step in determining peak height is illustrated in FIG. 43 where the signal 210 is shifted left or right to correspond with the position of expected peaks.
  • expected peaks such as expected peaks 212, 214, and 216.
  • putative peaks located in the signal such as putative peaks 218, 222, and 224 can be compared to the expected peaks 212, 214, and 216, respectively.
  • the entire signal is then shifted such that the putative peaks align more closely with the expected peaks.
  • the strongest putative peak is identified in FIG. 44.
  • the strongest peak is calculated as a combination of analyzing the overall peak height and area beneath the peak. For example, a moderately high but wide peak would be stronger than a very high peak that is extremely narrow.
  • a Gaussian 228 curve is fit to the peak 225. Once the Gaussian is fit, the width (W) of the Gaussian is determined and will be used as the peak width for future calculations.
  • the denoised, shifted, and baseline-corrected signal is not sufficiently processed for confidently calling the DNA composition of the biological sample.
  • the baseline has generally been removed, there are still residual baseline effects present. These residual baseline effects are therefore removed to increase the accuracy and confidence in making identifications.
  • FIG. 45 shows that the putative peaks 218, 222, and 224 are removed from the baseline corrected signal.
  • the peaks are removed by identifying a center line 230, 232, and 234 of the putative peaks 218, 222, and 224, respectively and removing an area to the left and to the right of the identified center line.
  • For each putative peak an area equal to twice the width (W) of the Gaussian is removed from the left of the center line, while an area equivalent to 50 daltons is removed from the right of the center line. It has been found that the area representing 50 daltons is adequate to sufficiently remove the effect of salt adducts which can be associated with an actual peak.
  • Such adducts appear to the right of an actual peak and are a natural effect from the chemistry involved in acquiring a mass spectrum. Although a 50 Dalton buffer has been selected, it will be appreciated that other ranges or methods can be used to reduce or eliminate adduct effects.
  • the peaks are removed and remaining minima 247 located as shown in FIG. 46 with the minima 247 connected to create signal 245.
  • a quartic polynomial is applied to signal 245 to generate a residual baseline 250 as shown in FIG. 47.
  • the residual baseline 250 is subtracted from the signal 225 to generate the final signal 255 as indicated in FIG. 48.
  • the residual baseline is the result of a quartic fit to signal 245, it will be appreciated that other techniques can be used to smooth or fit the residual baseline.
  • a Gaussian such as Gaussian 266, 268, and 270 is fit to each of the peaks, such as peaks 260, 262, and 264, respectively. Accordingly, the height of the Gaussian is determined as height 272, 274, and 276. Once the height of each Gaussian peak is determined, then the method of identifying a biological compound 35 can move into the genotyping phase 65 as shown in FIG. 25.
  • each putative peak is an actual peak. Accordingly, putative peaks with a strong signal-to-noise ratio are generally more likely to be an actual peak than a putative peak with a lower signal-to-noise ratio.
  • the height of each peak such as height 272, 274, and 276, is determined for each peak, with the height being an indicator of signal strength for each peak.
  • the noise profile such as noise profile 97, is extrapolated into noise profile 280 across the identified peaks. At the center line of each of the peaks, a noise value is determined, such as noise value 282, 283, and 284.
  • signal-to-noise ratios can be calculated for each peak. For example, the signal-to-noise ratio for the first peak in FIG. 50 would be calculated as signal value 272 divided by noise value 282, and in a similar manner the signal-to-noise ratio of the middle peak in FIG. 50 would be determined as signal 274 divided by noise value 283.
  • the signal-to-noise ratio is generally a useful indicator of the presence of an actual peak, further processing has been found to increase the confidence by which a sample can be identified.
  • the signal-to-noise ratio for each peak in the exemplarly embodiment can be adjusted by the goodness of fit between a Gaussian and each putative peak. It is a characteristic of a mass spectrometer that sample material is detected in a manner that generally complies with a normal distribution. Accordingly, greater confidence will be associated with a putative signal having a Gaussian shape than a signal that has a less normal distribution. The error resulting from having a non-Gaussian shape can be referred to as a “residual error”.
  • a residual error is calculated by taking a root mean square calculation between the Gaussian 293 and the putative peak 290 in the data signal. The calculation is performed on data within one width on either side of a center line of the Gaussian. The residual error is calculated as:
  • G is the Gaussian signal value
  • R is the putative peak value
  • N is the number of points from ⁇ W to +W.
  • An adjusted signal noise ratio is calculated for each putative peak using the formula (S/N) * EXP ( ⁇ 1 ⁇ R) , where S/N is the signal-to-noise ratio, and R is the residual error determined above.
  • S/N is the signal-to-noise ratio
  • R is the residual error determined above.
  • a probability is determined that a putative peak is an actual peak.
  • a probability profile 300 is generated where the adjusted signal-to-noise ratio is the x-axis and the probability is the y-axis. Probability is necessarily in the range between a 0% probability and a 100% probability, which is indicated as 1. Generally, the higher the adjusted signal-to-noise ratio, the greater the confidence that a putative peak is an actual peak.
  • the probability is 100% that the putative peak is an actual peak and can confidently be used to identify the DNA composition of a biological sample.
  • the target value of adjusted signal-to-noise ratio where the probability is assumed to be 100% is a variable parameter which is to be set according to application specific criteria. For example, the target signal-to-noise ratio will be adjusted depending upon trial experience, sample characteristics, and the acceptable error tolerance in the overall system. More specifically, for situations requiring a conservative approach where error cannot be tolerated, the target adjusted signal-to-noise ratio can be set to, for example, 10 and higher. Accordingly, 100% probability will not be assigned to a peak unless the adjusted signal-to-noise ratio is 10 or over.
  • the system can be set to assume a 100% probability with a 5 or greater target signal-to-noise ratio.
  • an intermediate signal-to-noise ratio target figure can be selected, such as 7, when a moderate risk of error can be assumed.
  • the allelic ratio between the signal strength of the highest peak and the signal strength of the second (or third and so on) highest peak should fall within an expected ratio. If the allelic ratio falls outside of normal guidelines, the exemplary embodiment imposes an allelic ratio penalty to the probability.
  • FIG. 53 shows an allelic penalty 315 which has an x-axis 317 that is the ratio between the signal strength of the second highest peak divided by signal strength of the highest peak. The yaxis 319 assigns a penalty between 0 and 1 depending on the determined allelic ratio.
  • allelic ratios over 30% are within the expected range and therefore no penalty is applied. Between a ratio of 10% and 30%, the penalty is linearly increased until at allelic ratios below 10% it is assumed the second-highest peak is not real.
  • the allelic penalty chart 315 is used to determine a penalty 319, which is multiplied by the peak probability determined in FIG. 52 to determine a final peak probability.
  • the statistical probability for various composition components can be determined, as an example, in order to determine the probability of each of three possible combinations of two peaks,—peak G, peak C and combinations GG, CC and GC.
  • FIG. 54 shows an example where a most probable peak 325 is determined to have a final peak probability of 90%. Peak 325 is positioned such that it represents a G component in the biological sample. Accordingly, it can be maintained that there is a 90% probability that G exists in the biological sample. Also in the example shown in FIG. 54, the second highest probability is peak 330 which has a peak probability of 20%. Peak 330 is at a position associated with a C composition. Accordingly, it can be maintained that there is a 20% probability that C exists in the biological sample.
  • the probability of combinations of G and C existing can be calculated.
  • FIG. 54 indicates that the probability of GG existing 329 is calculated as 72%. This is calculated as the probability of GG is equal to the probability of G existing (90%) multiplied by the probability of C not existing (100% ⁇ 20%). So if the probability of G existing is 90% and the probability of C not existing is 80%, the probability of GG is 72%.
  • the probability of CC existing is equivalent to the probability of C existing (20%) multiplied by the probability of G not existing (100% ⁇ 90%). As shown in FIG. 54, the probability of C existing is 20% while the probability of G not existing is 10%, so therefore the probability of CC is only 2%. Finally, the probability of GC existing is equal to the probability of G existing (90%) multiplied by the probability of C existing (20%). So if the probability of G existing is 90% and the probability of C existing is 20%, the probability of GC existing is 18%. In summary form, then, the probability of the composition of the biological sample is: probability of GG: 72%; probability of GC: 18%; and probability of CC: 2%.
  • FIG. 55 is used to decide whether or not sufficient confidence exists to call the genotype.
  • FIG. 55 shows a call chart 335 which has an x-axis 337 which is the ratio of the highest combination probability to the second highest combination probability.
  • the yaxis 339 simply indicates whether the ratio is sufficiently high to justify calling the genotype.
  • the value of the ratio can be indicated by M 340.
  • the value of M is set depending upon trial data, sample composition, and the ability to accept error. For example, the value M can be set relatively high, such as to a value 4 so that the highest probability must be at least four times greater than the second highest probability before confidence is established to call a genotype.
  • the value of M can be set to a more aggressive value, such as to 3, so that the ratio between the highest and second highest probabilities needs to be only a ratio of 3 or higher.
  • moderate value can be selected for M when a moderate risk can be accepted.
  • the probability of GG was 72% and the probability of GC was 18%
  • the ratio between 72% and 18% is 4.0, therefore, whether M is set to 3, 3.5, or 4, the system would call the genotype as GG.
  • the exemplary embodiment uses a ratio between the two highest peak probabilities to determine if a genotype confidently can be called, it will be appreciated that other methods can be substituted. It will also be appreciated that the above techniques can be used for calculating probabilities and choosing genotypes (or more general DNA patterns) containing of combinations of more than two peaks.
  • FIG. 56 a flow chart is shown generally defining the process of statistically calling genotype described above.
  • block 402 shows that the height of each peak is determined and that in block 404 a noise profile is extrapolated for each peak.
  • the signal is determined from the height of each peak in block 406 and the noise for each peak is determined using the noise profile in block 408.
  • block 410 the signal-to-noise ratio is calculated for each peak.
  • a residual error is determined in block 412 and an adjusted signal-to-noise ratio is calculated in block 414.
  • Block 416 shows that a probability profile is developed, with the probability of each peak existing found in block 418.
  • An allelic penalty can be applied in block 420, with the allelic penalty applied to the adjusted peak probability in block 422.
  • the probability of each combination of components is calculated in block 424 with the ratio between the two highest probabilities being determined in block 426. If the ratio of probabilities exceeds a threshold value then the genotype is called in block 428.
  • the computing device 20 supports “standardless” genotyping by identifying data peaks that contain putative SNPs.
  • Standardless genotyping is used, for example, where insufficient information is known about the samples to determine a distribution of expected peak locations, against which an allelic penalty as described above can be reliably calculated. This permits the computing device to be used for identification of peaks that contain putative SNPs from data generated by any assay that fragments a targeted DNA molecule.
  • peaks that are associated with an area under the data curve that deviates significantly from the typical area of other peaks in the data spectrum are identified and their corresponding mass (location along the x-axis) is determined.
  • peaks that deviate significantly from the average area of other peaks in the data are identified, and the expected allelic ratio between data peaks is defined in terms of the ratio of the area under the data peaks. Theoretically, where each genetic loci has the same molar concentration of analyte, the area under each corresponding peak should be the same, thus producing a 1.0 ratio of the peak area between any two peaks. In accordance with the methods provided herein, peaks having a smaller ratio relative to the other peaks in the data will not be recognized as peaks. More particularly, peaks having an area ratio smaller than 30% relative to a nominal value for peak area will be assigned an allelic penalty. The mass of the remaining peaks (their location along the x-axis of the data) will be determined based on oligonucleotide standards.
  • FIG. 57 shows a flow diagram representation of the processing by the computing device 20 (FIG. 24) when performing standardless genotyping.
  • the computing device receives data from the mass spectrometer.
  • the height of each putative peak in the data sample is determined, as indicated by the block 504.
  • a de-noise process 505 is performed, beginning with an extrapolation of the noise profile (block 506), followed by finding the noise of each peak (block 508) and calculating the signal to noise ratio for each data sample (block 510).
  • Each of these operations can be performed in accordance with the description above for denoise operations 45 of FIG. 25. Other suitable denoise operations will occur to those skilled in the art.
  • the next operation is to find the residual error associated with each data point. This is represented by the block 512 in FIG. 57.
  • the next step, block 514 involves calculating an adjusted signal to noise ratio for each identified peak.
  • a probability profile is developed next (block 516), followed by a determination of the peak probabilities at block 518.
  • the denoise operations of FIG. 57 comprising block 502 to block 518, comprise the corresponding operations described above in conjunction with FIG. 56 for block 402 through block 418, respectively.
  • the next action for the standardless genotype processing is to determine an allelic penalty for each peak, indicated by the block 524.
  • the standardless genotype processing of FIG. 57 determines an allelic penalty by comparing area under the peaks. Therefore, rather than compare signal strength ratios to determine an allelic penalty, such as described above for FIG. 53, the standardless processing determines the area under each of the identified peaks and compares the ratio of those areas. Determining the area under each peak can be computed using conventional numerical analysis techniques for calculating the area under a curve for experimental data.
  • the allelic penalty is assigned in accordance with FIG. 58, which shows that no penalty is assigned to peaks having a peak area relative to an expected average area value that is greater than 0.30 (30%).
  • the allelic penalty is applied to the peak probability value, which can be determined according to the process such as described in FIG. 52. It should be apparent from FIG. 58 that the allelic penalty imposed for peaks below a ratio of 30% is that such peaks will be removed from further measurement and processing. Other penalty schemes, however, can be imposed in accordance with knowledge about the data being processed, as determined by those skilled in the art.
  • the standardless genotype processing compares the location of the remaining putative peaks to oligonucleotide standards to determine corresponding masses in the processing for block 524.
  • the processing of the block 524 is performed to determine mass and genotype, rather than performing the operations corresponding to block 424, 426, and 428 of FIG. 33. Techniques for performing such comparisons and determining mass will be known to those skilled in the art.
  • the computing device 20 permits the detection and determination of the mass (location along the x-axis of the data) of the sense and antisense strand of fragments generated in the assay. If desired, the computing device can also detect and determine the quantity (area under each peak) of the respective sense and antisense strands, using a similar technique to that described above for standardless genotype processing. The data generated for each type of strand can then be combined to achieve a data redundancy and to thereby increase the confidence level of the determined genotype. This technique obviates primer peaks that are often observed in data from other diagnostic methods, thereby permitting a higher level of multiplexing. In addition, when quantitation is used in pooling experiments, the ratio of the measured peak areas is more reliably calculated than the peak identifying technique, due to data redundancy.
  • FIG. 23 is a flow diagram that illustrates the processing implemented by the computing device 20 to perform sense and antisense processing.
  • the computing device receives data from the mass spectrometer. This data will include data for the sense strand and antisense strand of assay fragments.
  • the height of each putative peak in the data sample is determined, as indicated by the block 604.
  • a de-noise process 605 is performed, beginning with an operation that extrapolates the noise profile (block 606), followed by finding the noise of each peak (block 608) and calculating the signal to noise ratio for each data sample (block 610).
  • the next operation is to find the residual error associated with each data point. This is represented by the block 612 in FIG. 36.
  • FIG. 23 shows that processing includes sense strand processing (block 630) and antisense strand processing (block 640).
  • Each block 630, 640 includes processing that corresponds to adjusting the signal to noise ratio, developing a probability profile, determining an allelic penalty, adjusting the peak probability by the allelic penalty, calculating genotype probabilities, and testing genotype probability ratios, such as described above in conjunction with blocks 414 through 426 of FIG. 56.
  • the processing of each block 630, 640 can, if desired, include standardless processing operations such as described above in conjunction with FIG. 57. The standardless processing can be included in place of or in addition to the processing operations of FIG. 56.
  • the data from the sense strand and antisense strand processing is combined and compared to expected database values to obtain the benefits of data redundancy as between the sense strand and antisense strand.
  • This processing is represented by the block 650.
  • the genotype processing is performed (block 660) and the genotype is identified.

Abstract

A high throughput method of determining frequencies of genetic variations is provided. The method includes steps of selecting a healthy target population and a genetic variation to be assessed; pooling a plurality of samples of biopolymers obtained from members of the population; determining or detecting the biopolymer that comprises the variation by mass spectrometry; obtaining a mass spectrum or a digital representation thereof; and determining the frequency of the variation in the population.

Description

    RELATED APPLICATIONS
  • This application is a divisional application of copending U.S. patent application Ser. No. 09/687,483, filed Oct. 13, 2000, to Andreas Braun, Hubert Koster, Dirk Van den Boom, Yip Ping, Charles Rodi, Liyan He, Norman Chiu and Christian Jurinke entitled “METHODS FOR GENERATING DATABASES AND DATABASES FOR IDENTIFYING POLYMORPHIC GENETIC MARKERS.”[0001]
  • Benefit of priority under 35 U.S.C. § 119(e) to the following provisional applications is claimed herein: [0002]
  • U.S. provisional application Serial No. 60/217,658 to Andreas Braun, Hubert Koster; Dirk Van den Boom, filed Jul. 10, 2000, entitled “METHODS FOR GENERATING DATABASES AND DATABASES FOR IDENTIFYING POLYMORPHIC GENETIC MARKERS”; U.S. provisional application Serial No. 60/159,176 to Andreas Braun, Hubert Koster, Dirk Van den Boom, filed Oct. 13, 1999, entitled “METHODS FOR GENERATING DATABASES AND DATABASES FOR IDENTIFYING POLYMORPHIC GENETIC MARKERS”; U.S. provisional application Serial No. 60/217,251, filed Jul. 10, 2000, to Andreas Braun, entitled “POLYMORPHIC KINASE ANCHOR PROTEIN GENE SEQUENCES, POLYMORPHIC KINASE ANCHOR PROTEINS AND METHODS OF DETECTING POLYMORPHIC KINASE ANCHOR PROTEINS AND NUCLEIC ACIDS ENCODING THE SAME”. This application is also a continuation-in-part of U.S. application Ser. No. 09/663,968, to Ping Yip, filed Sep. 19, 2000, entitled “METHOD AND DEVICE FOR IDENTIFYING A BIOLOGICAL SAMPLE.”[0003]
  • The above-noted applications and provisional applications are incorporated by reference in their entirety. [0004]
  • FIELD OF THE INVENTION
  • Process and methods for creating a database of genomic samples from healthy human donors. Methods that use the database to identify and correlate with polymorphic genetic markers and other markers with diseases and conditions are provided. [0005]
  • BACKGROUND
  • Diseases in all organisms have a genetic component, whether inherited or resulting from the body's response to environmental stresses, such as viruses and toxins. The ultimate goal of ongoing genomic research is to use this information to develop new ways to identify, treat and potentially cure these diseases. The first step has been to screen disease tissue and identify genomic changes at the level of individual samples. The identification of these “disease” markers has then fueled the development and commercialization of diagnostic tests that detect these errant genes or polymorphisms. With the increasing numbers of genetic markers, including single nucleotide polymorphisms (SNPs), microsatellites, tandem repeats, newly mapped introns and exons, the challenge to the medical and pharmaceutical communities is to identify genotypes which not only identify the disease but also follow the progression of the disease and are predictive of an organism's response to treatment. [0006]
  • Currently the pharmaceutical and biotechnology industries find a disease and then attempt to determine the genomic basis for the disease. This approach is time consuming and expensive and in many cases involves the investigator guessing as to what pathways might be involved in the disease. [0007]
  • Genomics [0008]
  • Presently the two main strategies employed in analyzing the available genomic information are the technology driven reverse genetics brute force strategy and the knowledge-based pathway oriented forward genetics strategy. The brute force approach yields large databases of sequence information but little information about the medical or other uses of the sequence information. Hence this strategy yields intangible products of questionable value. The knowledge-based strategy yields small databases that contain a lot of information about medical uses of particular DNA sequences and other products in the pathway and yield tangible products with a high value. [0009]
  • Polymorphisms [0010]
  • Polymorphisms have been known since 1901 with the identification of blood types. In the 1950's they were identified on the level of proteins using large population genetic studies. In the 1980's and 1990's many of the known protein polymorphisms were correlated with genetic loci on genomic DNA. For example, the gene dose of the [0011] apolipoprotein E type 4 allele was correlated with the risk of Alzheimer's disease in late onset families (see, e.g., Corder et al. (1993) Science 261: 921-923; mutation in blood coagulation factor V was associated with resistance to activated protein C (see, e.g., Bertina et aL. (1994) Nature 369:64-67); resistance to HIV-1 infection has been shown in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene (see, e.g., Samson et al. (1996) Nature 382:722-725); and a hypermutable tract in antigen presenting cells (APC, such as macrophages), has been identified in familial colorectal cancer in individuals of Ashkenzi jewish background (see, e.g., Laken et al. (1997) Nature Genet. 17:79-83). There can be more than three million polymorphic sites in the human genome. Many have been identified, but not yet characterized or mapped or associated with a marker.
  • Single Nucleotide Polymorphisms (SNPs) [0012]
  • Much of the focus of genomics has been in the identification of SNPs, which are important for a variety of reasons. They allow indirect testing (association of haplotypes) and direct testing (functional variants). They are the most abundant and stable genetic markers. Common diseases are best explained by common genetic alterations, and the natural variation in the human population aids in understanding disease, therapy and environmental interactions. [0013]
  • Currently, the only available method to identify SNPs in DNA is by sequencing, which is expensive, difficult and laborious. Furthermore, once a SNP is discovered it must be validated to determine if it is a real polymorphism and not a sequencing error. Also, discovered SNPs must then be evaluated to determine if they are associated with a particular phenotype. Thus, there is a need to develop new paradigms for identifying the genomic basis for disease and markers thereof. Therefore, it is an object herein to provide methods for identifying the genomic basis of disease and markers thereof. [0014]
  • SUMMARY
  • Databases and methods using the databases are provided herein. The databases comprise sets of parameters associated with subjects in populations selected only on the basis of being healthy (i e., where the subjects are mammals, such as humans, they are selected based upon apparent health and no detectable infections). The databases can be sorted based upon one or more of the selected parameters. [0015]
  • The databases, for example, can be relational databases, in which an index that represents each subject serves to relate parameters, which are the data, such as age, ethnicity, sex, medical history, etc. and ultimately genotypic information, that was inputted into and stored in the database. The database can then be sorted according to these parameters. Initially, the parameter information is obtained from a questionnaire answered by each subject from whom a body tissue or body fluid sample is obtained. As additional information about each sample is obtained, this information can be entered into the database and can serve as a sorting parameter. [0016]
  • The databases obtained from healthy individuals have numerous uses, such as correlating known polymorphisms with a phenotype or disease. The databases can be used to identify alleles that are deleterious, that are beneficial, and that are correlated with diseases. [0017]
  • For purposes herein, genotypic information can be obtained by any method known to those of skill in the art, but is generally obtained using mass spectrometry. [0018]
  • Also provided herein, is a new use for existing databases of subjects and genotypic and other parameters, such as age, ethnicity, race, and gender. Any database can be sorted according to the methods herein, and alleles that exhibit statistically significant correlations with any of the sorting parameters can be identified. It is noted, however, is noted, that the databases provided herein and randomly selected databases will perform better in these methods, since disease-based databases suffer numerous limitations, including their relatively small size, the homogeneity of the selected disease population, and the masking effect of the polymorphism associated with the markers for which the database was selected. Hence, the healthy database provided herein, provides advantages not heretofore recognized or exploited. The methods provided herein can be used with a selected database, including disease-based databases, with or without sorting for the discovery and correlation of polymorphisms. In addition, the databases provided herein represent a greater genetic diversity than the unselected databases typically utilized for the discovery of polymorphisms and thus allow for the enhanced discovery and correlation of polymorphisms. [0019]
  • The databases provided herein can be used for taking an identified polymorphism and ascertaining whether it changes in frequency when the data are sorted according to a selected parameter. [0020]
  • One use of these methods is correlating a selected marker with a particular parameter by following the occurrence of known genetic markers and then, having made this correlation, determining or identifying correlations with diseases. Examples of this use are p53 and Lipoprotein Lipase polymorphism. As exemplified herein, known markers are shown to have particular correlation with certain groups, such as a particular ethnicity or race or one sex. Such correlations will then permit development of better diagnostic tests and treatment regimens. [0021]
  • These methods are valuable for identifying one or more genetic markers whose frequency changes within the population as a function of age, ethnic group, sex or some other criteria. This can allow the identification of previously unknown polymorphisms and ultimately a gene or pathway involved in the onset and progression of disease. [0022]
  • The databases and methods provided herein permit, among other things, identification of components, particularly key components, of a disease process by understanding its genetic underpinnings and also permit an understanding of processes, such as individual drug responses. The databases and methods provided herein also can be used in methods involving elucidation of pathological pathways, in developing new diagnostic assays, identifying new potential drug targets, and in identifying new drug candidates. [0023]
  • The methods and databases can be used with experimental procedures, including, but are not limited to, in silico SNP identification, in vitro SNP identification/verification, genetic profiling of large populations, and in biostatistical analyses and interpretations. [0024]
  • Also provided herein, are combinations that contain a database provided herein and a biological sample from a subject in the database, and typically biological samples from all subjects or a plurality of subjects in the database. Collections of the tissue and body fluid samples are also provided. [0025]
  • Also, provided herein, are methods for determining a genetic marker that correlates with age, comprising identifying a polymorphism and determining the frequency of the polymorphism with increasing age in a healthy population. [0026]
  • Further provided herein are methods for determining whether a genetic marker correlates with susceptibility to morbidity, early mortality, or morbidity and early mortality, comprising identifying a polymorphism and determining the frequency of the polymorphism with increasing age in a healthy population. [0027]
  • Any of the methods herein described can be used out in a multiplex format. [0028]
  • Also provided are an apparatus and process for accurately identifying genetic information. It is another object herein that genetic information be extracted from genetic data in a highly automated manner. Therefore, to overcome the deficiencies in the known conventional systems, methods and apparatus for identifying a biological sample are provided. [0029]
  • Briefly, the method and system for identifying a biological sample generates a data set indicative of the composition of the biological sample. In a particular example, the data set is DNA spectrometry data received from a mass spectrometer. The data set is denoised, and a baseline is deleted. Since possible compositions of the biological sample can be known, expected peak areas can be determined. Using the expected peak areas, a residual baseline is generated to further correct the data set. Probable peaks are then identifiable in the corrected data set, which are used to identify the composition of the biological sample. In a disclosed example, statistical methods are employed to determine the probability that a probable peak is an actual peak, not an actual peak, or that the data too inconclusive to call. [0030]
  • Advantageously, the method and system for identifying a biological sample accurately makes composition calls in a highly automated manner. In such a manner, complete SNP profile information, for example, can be collected efficiently. More importantly, the collected data are analyzed with highly accurate results. For example, when a particular composition is called, the result can be relied upon with great confidence. Such confidence is provided by the robust computational process employed[0031]
  • DESCRIPTION OF THE DRAWINGS
  • FIG. 1 depicts an exemplary sample bank. [0032] Panel 1 shows the samples as a function of sex and ethnicity. Panel 2 shows the Caucasians as a function of age. Panel 3 shows the Hispanics as a function of age.
  • FIGS. 2A and 2C show an age- and sex-distribution of the 291S allele of the lipoprotein lipase gene in which a total of 436 males and 589 females were investigated. FIG. 2B shows an age distribution for the 436 males. [0033]
  • FIG. 3 is an exemplary questionnaire for population-based sample banking. [0034]
  • FIG. 4 depicts processing and tracking of blood sample components. [0035]
  • FIG. 5 depicts the allelic frequency of “sick” alleles and “healthy” alleles as a function of age. It is noted that the relative frequency of healthy alleles increases in a population with increasing age. [0036]
  • FIG. 6 depicts the age-dependent distribution of ApoE genotypes (see, Schächter et al. (1994) [0037] Nature Genetics 6:29-32).
  • FIG. 7A-D depicts age-related and genotype frequency of the p53 (tumor suppressor) [0038] codon 72 among the Caucasian population in the database. *R72 and *P72 represent the frequency of the allele in the database population. R72, R72P, and P72 represent the genotypes of the individuals in the population. The frequency of the homozygous P72 allele drops from 6.7% to 3.7% with age.
  • FIG. 8 depicts the allele and genotype frequencies of the p21 S31R allele as a function of age. [0039]
  • FIG. 9 depicts the frequency of the [0040] FVII Allele 353Q in pooled versus individual samples.
  • FIG. 10 depicts the frequency of the CETP (cholesterol ester transfer protein) allele in pooled versus individual samples. [0041]
  • FIG. 11 depicts the frequency of the plasminogen activator inhibitor-1 (PAI-1) 5G in pooled versus individual samples. [0042]
  • FIG. 12 shows mass spectra of the samples and the ethnic diversity of the PAI-1 alleles. [0043]
  • FIG. 13 shows mass spectra of the samples and the ethnic diversity of the CETP 405 alleles. [0044]
  • FIG. 14 shows mass spectra of the samples and the ethnic diversity of the Factor VII 353 alleles. [0045]
  • FIG. 15 shows ethnic diversity of PAI-1, CETP and Factor VII using the pooled DNA samples. [0046]
  • FIG. 16 shows the p53-Rb pathway and the relationships among the various factors in the pathway. [0047]
  • FIG. 17, which is a block diagram of a computer constructed to provide and process the databases described herein, depicts a typical computer system for storing and sorting the databases provided herein and practicing the methods provided herein. [0048]
  • FIG. 18 is a flow diagram that illustrates the processing steps performed using the computer illustrated in FIG. 17, to maintain and provide access to the databases for identifying polymorphic genetic markers. [0049]
  • FIG. 19 is a histogram showing the allele and genotype distribution in the age and sex stratified Caucasian population for the AKAP10-1 locus. Bright green bars show frequencies in individuals younger than 40 years. Dark green bars show frequencies in individuals older than 60 years. [0050]
  • FIG. 20 is a histogram showing the allele and genotype distribution in the age and sex stratified Caucasian population for the AKAP10-5 locus. Bright green bars show frequencies in individuals younger than 40 years; dark green bars show frequencies in individuals older than 60 years. [0051]
  • FIG. 21 is a histogram showing the allele and genotype distribution in the age and sex stratified Caucasian population for the h-msrA locus. Genotype difference between male age groups is significant. Bright green bars show frequencies in individuals younger than 40 years. Dark green bars show frequencies in individuals older than 60 years. [0052]
  • FIG. 22A-D is a sample data collection questionnaire used for the healthy database. [0053]
  • FIG. 23 is a flowchart showing processing performed by the computing device of FIG. 24 when performing genotyping of sense strands and antisense strands from assay fragments. [0054]
  • FIG. 24 is a block diagram showing a system provided herein; [0055]
  • FIG. 25 is a flowchart of a method of identifying a biological sample provided herein; [0056]
  • FIG. 26 is a graphical representation of data from a mass spectrometer; [0057]
  • FIG. 27 is a diagram of wavelet transformation of mass spectrometry data; [0058]
  • FIG. 28 is a graphical representation of [0059] wavelet stage 0 hi data;
  • FIG. 29 is a graphical representation of [0060] stage 0 noise profile;
  • FIG. 30 is a graphical representation of generating stage noise standard deviations; [0061]
  • FIG. 31 is a graphical representation of applying a threshold to data stages; [0062]
  • FIG. 32 is a graphical representation of a sparse data set; [0063]
  • FIG. 33 is a formula for signal shifting; [0064]
  • FIG. 34 is a graphical representation of a wavelet transformation of a denoised and shifted signal; [0065]
  • FIG. 35 is a graphical representation of a denoised and shifted signal; [0066]
  • FIG. 36 is a graphical representation of removing peak sections; [0067]
  • FIG. 37 is a graphical representation of generating a peak free signal; [0068]
  • FIG. 38 is a block diagram of a method of generating a baseline correction; [0069]
  • FIG. 39 is a graphical representation of a baseline and signal; [0070]
  • FIG. 40 is a graphical representation of a signal with baseline removed; [0071]
  • FIG. 41 is a table showing compressed data; [0072]
  • FIG. 42 is a flowchart of method for compressing data; [0073]
  • FIG. 43 is a graphical representation of mass shifting; [0074]
  • FIG. 44 is a graphical representation of determining peak width; [0075]
  • FIG. 45 is a graphical representation of removing peaks; [0076]
  • FIG. 46 is a graphical representation of a signal with peaks removed; [0077]
  • FIG. 47 is a graphical representation of a residual baseline; [0078]
  • FIG. 48 is a graphical representation of a signal with residual baseline removed; [0079]
  • FIG. 49 is a graphical representation of determining peak height; [0080]
  • FIG. 50 is a graphical representation of determining signal-to-noise for each peak; [0081]
  • FIG. 51 is a graphical representation of determining a residual error for each peak; [0082]
  • FIG. 52 is a graphical representation of peak probabilities; [0083]
  • FIG. 53 is a graphical representation of applying an allelic ratio to peak probability; [0084]
  • FIG. 54 is a graphical representation of determining peak probability; [0085]
  • FIG. 55 is a graphical representation of calling a genotype; [0086]
  • FIG. 56 is a flowchart showing a statistical procedure for calling a genotype; [0087]
  • FIG. 57 is a flowchart showing processing performed by the computing device of FIG. 1 when performing standardless genotyping; and [0088]
  • FIG. 58 is graphical representation of applying an allelic ratio to peak probability for standardless genotype processing.[0089]
  • DETAILED DESCRIPTION
  • Definitions [0090]
  • Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications and sequences from GenBank and other databases referred to herein throughout the disclosure are incorporated by reference in their entirety. [0091]
  • As used herein, a biopolymer includes, but is not limited to, nucleic acid, proteins, polysaccharides, lipids and other macromolecules. Nucleic acids include DNA, RNA, and fragments thereof. Nucleic acids can be derived from genomic DNA, RNA, mitochondrial nucleic acid, chloroplast nucleic acid and other organelles with separate genetic material. [0092]
  • As used herein, morbidity refers to conditions, such as diseases or disorders, that compromise the health and well-being of an organism, such as an animal. Morbidity susceptibility or morbidity-associated genes are genes that, when altered, for example, by a variation in nucleotide sequence, facilitate the expression of a specific disease clinical phenotype. Thus, morbidity susceptibility genes have the potential, upon alteration, of increasing the likelihood or general risk that an organism will develop a specific disease. [0093]
  • As used herein, mortality refers to the statistical likelihood that an organism, particularly an animal, will not survive a full predicted lifespan. Hence, a trait or a marker, such as a polymorphism, associated with increased mortality is observed at a lower frequency in older than younger segments of a population. [0094]
  • As used herein, a polymorphism, e.g. genetic variation, refers to a variation in the sequence of a gene in the genome amongst a population, such as allelic variations and other variations that arise or are observed. Thus, a polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. These differences can occur in coding and non-coding portions of the genome, and can be manifested or detected as differences in nucleic acid sequences, gene expression, including, for example transcription, processing, translation, transport, protein processing, trafficking, DNA synthesis, expressed proteins, other gene products or products of biochemical pathways or in post-translational modifications and any other differences manifested amongst members of a population. A single nucleotide polymorphism (SNP) refers to a polymorphism that arises as the result of a single base change, such as an insertion, deletion or change in a base. [0095]
  • A polymorphic marker or site is the locus at which divergence occurs. Such site can be as small as one base pair (an SNP). Polymorphic markers include, but are not limited to, restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats and other repeating patterns, simple sequence repeats and insertional elements, such as Alu. Polymorphic forms also are manifested as different mendelian alleles for a gene. Polymorphisms can be observed by differences in proteins, protein modifications, RNA expression modification, DNA and RNA methylation, regulatory factors that alter gene expression and DNA replication, and any other manifestation of alterations in genomic nucleic acid or organelle nucleic acids. [0096]
  • As used herein, a healthy population refers to a population of organisms, including but are not limited to, animals, bacteria, viruses, parasites, plants, eubacteria, and others, that are disease free. The concept of disease-free is a function of the selected organism. For example, for mammals it refers to a subject not manifesting any disease state. Practically a healthy subject, when human, is defined as human donor who passes blood bank criteria to donate blood for eventual use in the general population. These criteria are as follows: free of detectable viral, bacterial, mycoplasma, and parasitic infections; not anemic; and then further selected based upon a questionnaire regarding history (see FIG. 3). Thus, a healthy population represents an unbiased population of sufficient health to donate blood according to blood bank criteria, and not further selected for any disease state. Typically such individuals are not taking any medications. For plants, for example, it is a plant population that does not manifest diseases pathology associated with plants. For bacteria it is a bacterial population replicating without environmental stress, such as selective agents, heat and other pathogens. [0097]
  • As used herein, a healthy database (or healthy patient database) refers to a database of profiles of subjects that have not been pre-selected for any particular disease. Hence, the subjects that serve as the source of data for the database are selected, according to predetermined criteria, to be healthy. In contrast to other such databases that have been pre-selected for subjects with a particular disease or other characteristic, the subjects for the database provided herein are not so-selected. Also, if the subjects do manifest a disease or other condition, any polymorphism discovered or characterized should be related to an independent disease or condition. In a one embodiment, where the subjects are human, a healthy subject manifests no disease symptoms and meets criteria, such as those set by blood banks for blood donors. [0098]
  • Thus, the subjects for the database are a population of any organism, including, but are not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity that has nucleic acid. Among subjects are mammals, such as, although not necessarily, humans. Such a database can capture the diversity of a population, thus providing for discovery of rare polymorphisms. [0099]
  • As used herein, a profile refers to information relating to, but not limited to and not necessarily including all of, age, sex, ethnicity, disease history, family history, phenotypic characteristics, such as height and weight and other relevant parameters. A sample collect information form is shown in FIG. 22, which illustrates profile intent. [0100]
  • As used herein, a disease state is a condition or abnormality or disorder that can be inherited or result from environmental stresses, such as toxins, bacterial, fungal and viral infections. [0101]
  • As used herein, set of non-selected subjects means that the subjects have not been pre-selected to share a common disease or other characteristic. They can be selected to be healthy as defined herein. [0102]
  • As used herein, a phenotype refers to a set of parameters that includes any distinguishable trait of an organism. A phenotype can be physical traits and can be, in instances in which the subject is an animal, a mental trait, such as emotional traits. Some phenotypes can be determined by observation elicited by questionnaires (see, e.g., FIGS. [0103] 3 and 22) or by referring to prior medical and other records. For purposes herein, a phenotype is a parameter around which the database can be sorted.
  • As used herein, a parameter is any input data that will serve as a basis for sorting the database. These parameters will include phenotypic traits, medical histories, family histories and any other such information elicited from a subject or observed about the subject. A parameter can describe the subject, some historical or current environmental or social influence experienced by the subject, or a condition or environmental influence on someone related to the subject. Paramaters include, but are not limited to, any of those described herein, and known to those of skill in the art. [0104]
  • As used herein, haplotype refers to two or polymorphism located on a single DNA strand. Hence, haplotyping refers to identification of two or more polymorphisms on a single DNA strand. Haplotypes can be indicative of a phenotype. For some disorders a single polymorphism can suffice to indicate a trait; for others a plurality (i.e., a haplotype) can be needed. Haplotyping can be performed by isolating nucleic acid and separating the strands. In addition, when using enzymes such a certain nucleases, that produce, different size fragments from each strand, strand separation is not needed for haplotyping. [0105]
  • As used herein, pattern with reference to a mass spectrum or mass spectrometric analyses, refers to a characteristic distribution and number of signals (such peaks or digital representations thereof). [0106]
  • As used herein, signal in the context of a mass spectrum and analysis thereof refers to the output data, which the number or relative number of moleucles having a particular mass. Signals include “peaks” and digital representations thereof. [0107]
  • As used herein, adaptor, when used with reference to haplotyping using Fen ligase, refers to a nucleic acid that specifically hybridizes to a polymorphism of interest. An adaptor can be partially double-stranded. An adaptor complex is formed when an adaptor hybridizes to its target. [0108]
  • As used herein, a target nucleic acid refers to any nucleic acid of interest in a sample. It can contain one or more nucleotides. [0109]
  • As used herein, standardless analysis refers to a determination based upon an internal standard. For example, the frequency of a polymorphism can be determined herein by comparing signals within a single mass spectrum. [0110]
  • As used herein, amplifying refers to methods for increasing the amount of a bipolymer, especially nucleic acids. Based on the 5′ and 3′ primers that are chosen, amplication also serves to restrict and define the region of the genome which is subject to analysis. Amplification can be performed by any method known to those skilled in the art, including use of the polymerase chain reaction (PCR) etc. Amplification, e.g., PCR must be done quantitatively when the frequency of polymorphism is required to be determined. [0111]
  • As used herein, cleaving refers to non-specific and specific fragmentation of a biopolymer. [0112]
  • As used herein, multiplexing refers to the simultaneous detection of more than one polymorphism. Methods for performing multiplexed reactions, particularly in conjunction with mass spectrometry are known (see, e.g., U.S. Pat. Nos. 6,043,031, 5,547,835 and International PCT application No. WO 97/37041). [0113]
  • As used herein, reference to mass spectrometry encompasss any suitable mass spectrometric format known to those of skill in the art. Such formats iniude, but are not limited to, Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF), Electrospray (ES), IR-MALDI (see, e.g., published International PCT application No.99/57318 and U.S. Pat. No. 5,118,937), Ion Cyclotron Resonance (ICR), Fourier Transform and combinations thereof. MALDI, particular UV and IR, are among the formats contemplated. [0114]
  • As used herein, mass spectrum refers to the presentation of data obtained from analyzing a biopolymer or fragment thereof by mass spectrometry either graphically or encoded numerically. [0115]
  • As used herein, a blood component is a component that is separated from blood and includes, but is not limited to red blood cells and platelets, blood clotting factors, plasma, enzymes, plasminogen, immunoglobulins. A cellular blood component is a component of blood, such as a red blood cell, that is a cell. A blood protein is a protein that is normally found in blood. Examples of such proteins are blood factors VII and VII. Such proteins and components are well-known to those of skill in the art. [0116]
  • As used herein, plasma can be prepared by any method known to those of skill in the art. For example, it can be prepared by centrifuging blood at a force that pellets the red cells and forms an interface between the red cells and the buffy coat, which contains leukocytes, above which is the plasma. For example, typical platelet concentrates contain at least about 10% plasma. [0117]
  • Blood can be separated into its components, including, but not limited to, plasma, platelets and red blood cells by any method known to those of skill in the art. For example, blood can be centrifuged for a sufficient time and at a sufficient acceleration to form a pellet containing the red blood cells. Leukocytes collect primarily at the interface of the pellet and supernatant in the buffy coat region. The supernatant, which contains plasma, platelets, and other blood components, can then be removed and centrifuged at a higher acceleration, whereby the platelets pellet. [0118]
  • As used herein, p53 is a cell cycle control protein that assesses DNA damage and acts as a transcription factor regulation gene which control cell growth, DNA repair and apoptosis. The p53 mutations have been found in a wide variety of different cancers, including all of the different types of leukemia, with varying frequency. The loss of normal p53 functions results in genomic instability and uncontrolled growth of the host cell. [0119]
  • As used herein, p21 is a cyclin-dependent kinase inhibitor, associated with G1 phase arrest of normal cells. Expression triggers apoptosis or programmed cell death and has been associated with Wilms' tumor, a pediatric kidney cancer. [0120]
  • As used herein, Factor VII is a serine protease involved the extrinsic blood coagulation cascade. This factor is activated by thrombin and works with tissue factor (Factor III) in the processing of Factor X to Factor Xa. Evidence has supported an association between polymorphisms in the gene and increase Factor VII activity which can result in an elevated risk of ischemic cardiovascular disease including myocardial infarction. [0121]
  • As used herein, a relational database stores information in a form representative of matrices, such as two-dimensional tables, including rows and columns of data, or higher dimensional matrices. For example, in one embodiment, the relational database has separate tables each with a parameter. The tables are linked with a record number, which also acts as an index. The database can be searched or sorted by using data in the tables and is stored in any suitable storage medium, such as floppy disk, CD rom disk, hard drive or other suitable medium. [0122]
  • As used herein, a bar codes refers any array of optically readable marks of any desired size and shape that are arranged in a reference context or frame of, typically, although not necessarily, one or more columns and one or more rows. For purposes herein, the bar code refers to any symbology, not necessary “bar” but can include dots, characters or any symbol or symbols. [0123]
  • As used herein, symbology refers to an identifier code or symbol, such as a bar code, that is linked to a sample. The index will reference each such symbology. The symbology is any code known or designed by the user. The symbols are associated with information stored in the database. For example, each sample can be uniquely identified with an encoded symbology. The parameters, such as the answers to the questions and subsequent genotypic and other information obtained upon analysis of the samples is included in the database and associated with the symbology. The database is stored on any suitable recording medium, such as a hard drive, a floppy disk, a tape, a CD ROM, a DVD disk and any other suitable medium. [0124]
  • Databases [0125]
  • Human genotyping is currently dependent on collaborations with hospitals, tissues banks and research institutions that provide samples of disease tissue. This approach is based on the concept that the onset and/or progression of diseases can be correlated with the presence of a polymorphisms or other genetic markers. This approach does not consider that disease correlated with the presence of specific markers and the absence of specific markers. It is shown herein that identification and scoring of the appearance and disappearance of markers is possible only if these markers are measured in the background of healthy subjects where the onset of disease does not mask the change in polymorphism occurrence. Databases of information from disease populations suffer from small sample size, selection bias and heterogeneity. The databases provided herein from healthy populations solve these problems by permitting large sample bands, simple selection methods and diluted heterogeneity. [0126]
  • Provided herein are first databases of parameters, associated with non-selected, particularly healthy, subjects. Also provided are combinations of the databases with indexed samples obtained from each of the subjects. Further provided are databases produced from the first databases. These contain, in addition to the original parameters, information, such as genotypic information, including, but are not limited to, genomic sequence information, derived from the samples. [0127]
  • The databases, which are herein designated healthy databases, are so-designated because they are not obtained from subjects pre-selected for a particular disease. Hence, although individual members can have a disease, the collection of individuals is not selected to have a particular disease. [0128]
  • The subjects from whom the parameters are obtained comprise either a set of subjects who are randomly selected across, typically, all populations, or are pre-selected to be disease-free or healthy. As a result, the database is not selected to be representative of any pre-selected phenotype, genotype, disease or other characteristic. Typically the number of subjects from which the database is prepared is selected to produce statistically significant results when used in the methods provided herein. Generally, the number of subjects will be greater than 100, 200, and typically than 1000. The precise number can be empirically determined based upon the frequency of the parameter(s) that can be used to sort the database. Generally the population can have at least 50, at least 100, at least 200, at least 500, at least 1000, at least 5000 or at least 10,000 or more subjects. [0129]
  • Upon identification of a collection of subjects, information about each subject is recorded and associated with each subject as a database. The information associated with each of the subjects, includes, but is not limited to, information related to historical characteristics of the subjects, phenotypic characteristics and also genotypic characteristics, medical characteristics and any other traits and characteristics about the subject that can be determined. This information will serve as the basis for sorting the database. [0130]
  • In an exemplary embodiment, the subjects are mammals, such as humans, and the information relates to one or more of parameters, such as age, sex, medical history, ethnicity and any other factor. Such information, when the animals are humans, for example, can be obtained by a questionnaire and by observations about the individual, such as hair color, eye color and other characteristics. Genotypic information can be obtained from tissue or other body and body fluid samples from the subject. [0131]
  • The healthy genomic database can include profiles and polymorphisms from healthy individuals from a library of blood samples where each sample in the library is an individual and separate blood or other tissue sample. Each sample in the database is profiled as to the sex, age, ethnic group, and disease history of the donor. [0132]
  • The databases are generated by first identifying healthy populations of subjects and obtaining information about each subject that will serve as the sorting parameters for the database. This information can be entered into a storage medium, such as the memory of a computer. [0133]
  • The information obtained about each subject in a population used for generating the database is stored in a computer memory or other suitable storage medium. The information is linked to an identifier associated with each subject. Hence the database will identify a subject, for example by a datapoint representative of a bar code, and then all information, such as the information from a questionnaire, regarding the individual is associated with the datapoint. As the information is collected the database is generated. [0134]
  • Thus, for example, profile information, such as subject histories obtained from questionnaires, is collected in the database. The resulting database can be sorted as desired, using standard software, such as by age, sex and/or ethnicity. An exemplary questionnaire for subjects from whom samples are to be obtained is shown in FIGS. [0135] 22A-D. Each questionnaire, for example, can be identified by a bar code, particularly a machine readable bar code for entry into the database. After a subject provides data and is deemed to be healthy (i.e., meets standards for blood donation), the data in the questionnaire is entered into the database and is associated with the bar code. A tissue, cell or blood sample is obtained from the subject.
  • FIG. 4 exemplifies processing and tracking of blood sample components. Each component is tracked with a bar code, dated, is entered into the database and associated with the subject and the profile of the subject. Typically, the whole blood is centrifuged to produce plasma, red blood cells (which pellet) and leukocytes found in the buffy coat which layers in between. Various samples are obtained and coded with a bar code and stored for use as needed. [0136]
  • Samples are collected from the subjects. The samples include, but are not limited to, tissues, cells, and fluids, such as nucleic acid, blood, plasma, amniotic fluid, synovial fluid, urine, saliva, aqueous humor, sweat, sperm samples and cerebral spinal fluid. It is understood that the particular set of samples depends upon the organisms in the population. [0137]
  • Once samples are obtained the collection can be stored and, in some embodiments, each sample is indexed with an identifier, particularly a machine readable code, such as a bar code. For analyses, the samples or components of the samples, particularly biopolymers and small molecules, such as nucleic acids and/or proteins and metabolites, are isolated. [0138]
  • After samples are analyzed, this information is entered into the database in the memory of the storage medium and associated with each subject. This information includes, but is not limited to, genotypic information. Particularly, nucleic acid sequence information and other information indicative of polymorphisms, such as masses of PCR fragments, peptide fragment sequences or masses, spectra of biopolymers and small molecules and other indicia of the structure or function of a gene, gene product or other marker from which the existence of a polymorphism within the population can be inferred. [0139]
  • In an exemplary embodiment, a database can be derived from a collection of blood samples. For example, FIG. 1(see, also FIG. 10) shows the status of a collection of over 5000 individual samples. The samples were processed in the laboratory following SOP (standard operating procedure) guidelines. Any standard blood processing protocol can be used. [0140]
  • For the exemplary database described herein, the following criteria were used to select subjects: [0141]
  • No testing is done for infectious agents. [0142]
  • Age: At least 17 years old [0143]
  • Weight: Minimum of 110 pounds [0144]
  • Permanently Disqualified: [0145]
  • History of hepatitis (after age 11) [0146]
  • Leukemia Lymphoma [0147]
  • Human immunodeficiency virus (HIV),AIDS [0148]
  • Chronic kidney disease [0149]
  • Temporarily Disqualified: [0150]
  • Pregnancy—until six weeks after delivery, miscarriage or abortion [0151]
  • Major surgery or transfusions—for one year [0152]
  • Mononucleosis—until complete recovery [0153]
  • Prior whole blood donation—for eight weeks [0154]
  • Antibiotics by injection for one week; by mouth, for forty-eight hours, except antibiotics for skin complexion; [0155]
  • 5 year Deferment: [0156]
  • Internal cancer and skin cancer if it has been removed, is healed and there is no recurrence [0157]
  • These correspond to blood bank criteria for donating blood and represent a healthy population as defined herein for a human healthy database. [0158]
  • 5 Structure of the Database [0159]
  • Any suitable database structure and format known to those of skill in the art can be employed. For example, a relational database is a an exemplary format in which data are stored as matrices or tables of the parameters linked by an indexer that identifies each subject. Software for preparing and manipulating, including sorting the database, can be readily developed or adapted from commercially available software, such as Microsoft Access. [0160]
  • Quality Control [0161]
  • Quality control procedures can be implemented. For example, after collection of samples, the quality of the collection in the bank can be assessed. For example, mix-up of samples can be checked by testing for known markers, such as sex. After samples are separated by ethnicity, samples are randomly tested for a marker associated with a particular ethnicity, such as HLA DQA1 group specific component, to assess whether the samples have been properly sorted by ethnic group. An exemplary sample bank is depicted in FIG. 4. [0162]
  • Obtaining Genotypic Data and Other Parameters for the Database [0163]
  • After informational and historical parameters are entered into the database, material from samples obtained from each subject, is analyzed. Analyzed material include proteins, metabolites, nucleic acids, lipids and any other desired constituent of the material. For example, nucleic acids, such as genomic DNA, can be analyzed by sequencing. [0164]
  • Sequencing can be performed using any method known to those of skill in the art. For example, if a polymorphism is identified or known, and it is desired to assess its frequency or presence among the subjects in the database, the region of interest from each sample can be isolated, such as by PCR or restriction fragments, hybridization or other suitable method known to those of skill in the art and sequenced. For purposes herein, sequencing analysis can be effected using mass spectrometry (see, e.g., U.S. Pat. Nos. 5,547,835, 5,622,824, 5,851,765, and 5,928,906). Nucleic acids also can be sequenced by hybridization (see, e.g., U.S. Pat. Nos. 5,503,980, 5,631,134, 5,795,714) and including analysis by mass spectrometry (see, U.S. application Ser. Nos. 08/419,994 and 09/395,409). [0165]
  • In other detection methods, it is necessary to first amplify prior to identifying the allelic variant. Amplification can be performed, e.g., by PCR and/or LCR, according to methods known in the art. In one embodiment, genomic DNA of a cell is exposed to two PCR primers and amplification for a number of cycles sufficient to produce the required amount of amplified DNA. In some embodiments, the primers are located between 150 and 350 base pairs apart. [0166]
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. [0167]
  • Nucleic acids also can be analyzed by detection methods and protocols, particularly those that rely on mass spectrometry (see, e.g., U.S. Pat. No. 5,605,798, 6,043,031, allowed copending U.S. application Ser. No. 08/744,481, U.S. application Ser. No. 08/990,851 and International PCT application No. WO 99/31278, International PCT application No. WO 98/20019). These methods can be automated (see, e.g., copending U.S. application Ser. No. 09/285,481 and published International PCT application No. PCT/US00/08111, which describes an automated process line). Among the methods of analysis herein are those involving the primer oligo base extension (PROBE) reaction with mass spectrometry for detection (described herein and elsewhere, see e.g., U.S. Pat. No. 6,043,031; see, also U.S. application Ser. Nos. 09/287,681, 09/287,682, 09/287,141 and 09/287,679, allowed copending U.S. application Ser. No. 08/744,481, International PCT application No. PCT/US97/20444, published as International PCT application No. WO 98/20019, and based upon U.S. application Ser. Nos. 08/744,481, 08/744,590, 08/746,036, 08/746,055, 08/786,988, 08/787,639, 08/933,792, 08/746,055, 08/786,988 and 08/787,639; see, also U.S. application Ser. No. 09/074,936, U.S. Pat. No. 6,024,925, and U.S. application Ser. Nos. 08/746,055 and 08/786,988, and published International PCT application No. WO 98/20020) [0168]
  • A chip based format in which the biopolymer is linked to a solid support, such as a silicon or silicon-coated substrate, such as in the form of an array, is among the formats for performing the analyses is. Generally, when analyses are performed using mass spectrometry, particularly MALDI, small nanoliter volumes of sample are loaded on, such that the resulting spot is about, or smaller than, the size of the laser spot. It has been found that when this is achieved, the results from the mass spectrometric analysis are quantitative. The area under the signals in the resulting mass spectra are proportional to concentration (when normalized and corrected for background). Methods for preparing and using such chips are described in U.S. Pat. No. 6,024,925, co-pending U.S. application Ser. Nos. 08/786,988, 09/364,774, 09/371,150 and 09/297,575; see, also U.S. application Ser. No. PCT/US97/20195, which published as WO 98/20020. Chips and kits for performing these analyses are commercially available from SEQUENOM under the trademark MassARRAY. MassArray relies on the fidelity of the enzymatic primer extension reactions combined with the miniaturized array and MALDI-TOF (Matrix-Assisted Laser Desorption Ionization-Time of Flight) mass spectrometry to deliver results rapidly. It accurately distinguishes single base changes in the size of DNA fragments associated with genetic variants without tags. [0169]
  • The methods provided herein permit quantitative determination of alleles. The areas under the signals in the mass spectra can be used for quantitative determinations. The frequency is determined from the ratio of the signal to the total area of all of the spectrum and corrected for background. This is possible because of the PROBE technology as described in the above applications incorporated by reference herein. [0170]
  • Additional methods of analyzing nucleic acids include amplification-based methods including polymerase chain reaction (PCR), ligase chain reaction (LCR), mini-PCR, rolling circle amplification, autocatalytic methods, such as those using Qβ replicase, TAS, 3SR, and any other suitable method known to those of skill in the art. [0171]
  • Other methods for analysis and identification and detection of polymorphisms, include but are not limited to, allele specific probes, Southern analyses, and other such analyses. [0172]
  • The methods described below provide ways to fragment given amplified or non-amplified nucleotide sequences thereby producing a set of mass signals when mass spectrometry is used to analyze the fragment mixtures. [0173]
  • Amplified fragments are yielded by standard polymerase chain methods (U.S. Pat. Nos. 4,683,195 and 4,683,202). The fragmentation method involves the use of enzymes that cleave single or double strands of DNA and enzymes that ligate DNA. The cleavage enzymes can be glycosylases, nickases, and site-specific and non site-specific nucleases, such as, but are not limited to, glycosylases, nickases and site-specific nucleases. [0174]
  • Glycosylase Fragmentation Method [0175]
  • DNA glycosylases specifically remove a certain type of nucleobase from a given DNA fragment. These enzymes can thereby produce abasic sites, which can be recognized either by another cleavage enzyme, cleaving the exposed phosphate backbone specifically at the abasic site and producing a set of nucleobase specific fragments indicative of the sequence, or by chemical means, such as alkaline solutions and or heat. The use of one combination of a DNA glycosylase and its targeted nucleotide would be sufficient to generate a base specific signature pattern of any given target region. [0176]
  • Numerous DNA gicosylases are known, For example, a DNA glycosylase can be uracil-DNA glycolsylase (UDG) , 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, thymine mismatch-DNA glycosylase, hypoxanthine-DNA glycosylase, 5-Hydroxymethyluracil DNA glycosylase (HmUDG), 5-Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenine DNA glycosylase (see, e.g.,, U.S. Pat. Nos. 5,536,649, 5,888, 795, 5,952,176 and 6,099,553, International PCT application Nos. WO 97/03210, WO 99/54501; see, also, Eftedal et al. (1993) Nucleic Acids Res 21:2095-2101, Bjelland and Seeberg (1987) Nucleic Acids Res. 15:2787-2801, Saparbaev et al. (1995) Nucleic Acids Res. 23:3750-3755, Bessho (1999) Nucleic Acids Res. 27:979-983) corresponding to the enzyme's modified nucleotide or nucleotide analog target. uracil-DNA glycolsylase (UDG) is an exemplary glycosylase. [0177]
  • Uracil, for example, can be incorporated into an amplified DNA molecule by amplifying the DNA in the presence of normal DNA precursor nucleotides (e.g. dCTP, dATP, and dGTP) and dUTP. When the amplified product is treated with UDG, uracil residues are cleaved. Subsequent chemical treatment of the products from the UDG reaction results in the cleavage of the phosphate backbone and the generation of nucleobase specific fragments. Moreover, the separation of the complementary strands of the amplified product prior to glycosylase treatment allows complementary patterns of fragmentation to be generated. Thus, the use of dUTP and Uracil DNA glycosylase allows the generation of T specific fragments for the complementary strands, thus providing information on the T as well as the A positions within a given sequence. Similar to this, a C-specific reaction on both (complementary) strands (i.e. with a C-specific glycosylase) yields information on C as well as G positions within a given sequence if the fragmentation patterns of both amplification strands are analyzed separately. Thus, with the glycosylase method and mass spectrometry, a full series of A, C, G and T specific fragmentation patterns can be analyzed. [0178]
  • Nickase Fragmentation Method [0179]
  • A DNA nickase, or DNase, can be used to recognize and cleave one strand of a DNA duplex. Numerous nickases are known. Among these, for example, are nickase NY2A nickase and NYS1 nickase (Megabase) with the following cleavage sites: [0180]
  • NY2A: 5′ . . . R AG . . . 3′[0181]
  • 3′ . . . Y TC . . . 5′ where R=A or G and Y=C or T [0182]
  • NYS1: 5′ . . . CC[A/G/T] . . . 3′[0183]
  • 3′ . . . GG[T/C/A] . . . 5′. [0184]
  • Fen-Ligase Fragmentation Method [0185]
  • The Fen-ligase method involves two enzymes: Fen-1 enzyme and a ligase. The Fen-1 enzyme is a site-specific nuclease known as a “flap” endonuclease (U.S. Pat. Nos. 5,843,669, 5,874,283, and 6,090,606). This enzyme recognizes and cleaves DNA “flaps” created by the overlap of two oligonucleotides hybridized to a target DNA strand. This cleavage is highly specific and can recognize single base pair mutations, permitting detection of a single homologue from an individual heterozygous at one SNP of interest and then genotyping that homologue at other SNPs occurring within the fragment. Fen-1 enzymes can be Fen-1 like nucleases e.g. human, murine, and Xenopus XPG enzymes and yeast RAD2 nucleases or Fen-1 endonucleases from, for example, [0186] M. jannaschii, P. furiosus, and P. woesei. Among such enzymes are the Fen-1 enzymes.
  • The ligase enzyme forms a phosphodiester bond between two double stranded nucleic acid fragments. The ligase can be DNA Ligase I or DNA Ligase IlIl (see, e.g., U.S. Pat. Nos. 5,506,137, 5,700,672, 5,858,705 and 5,976,806; see, also, Waga, et al. (1994) J. Biol. Chem. 269:10923-10934, Li et al. (1994) Nucleic Acids Res. 22:632-638, Arrand et al. (1986) J. Biol. Chem. 261:9079-9082, Lehman (1974) Science 186:790-797, Higgins and Cozzarelli (1979) Methods Enzymol. 68:50-71, Lasko et al. (1990) Mutation Res. 236:277-287, and Lindahl and Barnes (1992) Ann. Rev. Biochem. 61:251-281 ). Thermostable ligase (Epicenter Technologies), where “thermostable” denotes that the ligase retains activity even after exposure to temperatures necessary to separate two strands of DNA, are among the ligases for use herein. [0187]
  • Type IIS Enzyme Fragmentation Method [0188]
  • Restriction enzymes bind specifically to and cleave double-stranded DNA at specific sites within or adjacent to a particular recognition sequence. These enzymes have been classified into three groups (e.g. Types I, II, and III) as known to those of skill in the art. Because of the properties of type I and type III enzymes, they have not been widely used in molecular biological applications. Thus, for purposes herein type II enzymes are among those contemplated. Of the thousands of restriction enzymes known in the art, there are 179 different type II specificities. Of the 179 unique type II restriction endonucleases, 31 have a 4-base recognition sequence, 11 have a 5-base recognition sequence, 127 have a 6-base recognition sequence, and 10 have recognition sequences of greater than six bases (U.S. Pat. No. 5,604,098). Of category type II enzymes, type IIS is exemplified herein. [0189]
  • Type IIS enzymes can be Alw XI, Bbv I, [0190] Bce 83, Bpm I, Bsg I, Bsm AI, Bsm FI, Bsa I, Bcc I, Bcg I, Ear I, Eco 57I, Esp 3I, Fau I, Fok I, Gsu I, Hga I, Mme I, Mbo II, Sap I, and the otheres.
  • The Fok I enzyme endonuclease is an exemplary well characterized member of the Type IIS class (see, e.g., U.S. Pat. Nos. 5,714,330, 5,604,098, 5,436,150, 6,054,276 and 5,871,911; see, also, Szybalski et al. (1991 ) Gene 100:13-26, Wilson and Murray (1991) Ann. Rev. Genet. 25:585-627, Sugisaki et al. (1981) Gene 16:73-78, Podhajska and Szalski (1985) Gene 40:175-182. Fok I recognizes the [0191] sequence 5′GGATG-3′ and cleaves DNA accordingly. Type IIS restriction sites can be introduced into DNA targets by incorporating the sites into primers used to amplify such targets. Fragments produced by digestion with Fok I are site specific and can be analyzed by mass spectrometry methods such as MALDI-TOF mass spectrometry, ESI-TOF mass spectrometry, and any other type of mass spectrometry well known to those of skill in the art.
  • Once a polymorphism has been found to correlate with a parameter such as age, age groups can be screened for polymorphisms. The possibility of false results due to allelic dropout is examined by doing comparative PCR in an adjacent region of the genome. [0192]
  • Analyses [0193]
  • In using the database, allelic frequencies can be determined across the population by analyzing each sample in the population individually, determining the presence or absence of allele or marker of interest in each individual sample, and then determining the frequency of the marker in the population. The database can then be sorted (stratified) to identify any correlations between the allele and a selected parameter using standard statistical analysis. If a correlation is observed, such as a decrease in a particular marker with age or correlation with sex or other parameter, then the marker is a candidate for further study, such as genetic mapping to identify a gene or pathway in which it is involved. The marker can then be correlated, for example, with a disease. Haplotying also can be carried out. Genetic mapping can be effected using standard methods and can also require use of databases of others, such as databases previously determined to be associated with a disorder. [0194]
  • Exemplary analyses have been performed and these are shown in the figures, and discussed herein. [0195]
  • Sample Pooling [0196]
  • It has been found that using the databases provided herein, or any other database of such information, substantially the same frequencies that were obtained by examining each sample separately can be obtained by pooling samples, such as in batches of 10, 20, 50, 100, 200, 500, 1000 or any other number. A precise number can be determined empirically if necessary, and can be as low as 3. [0197]
  • In one embodiment, the frequency of genotypic and other markers can be obtained by pooling samples. To do this a target population and a genetic variation to be assessed is selected, a plurality of samples of biopolymers are obtained from members of the population, and the biopolymer from which the marker or genotype can be inferred is determined or detected. A comparison of samples tested in pools and individually and the sorted results therefrom are shown in FIG. 9, which shows frequency of the [0198] factor VII Allele 353Q. FIG. 10 depicts the frequency of the CETP Allele in pooled versus individual samples. FIG. 15 shows ethnic diversity among various ethnic groups in the database using pooled DNA samples to obtain the data. FIGS. 12-14 show mass spectra for these samples.
  • Pooling of test samples has application not only to the healthy databases provided herein, but also to use in gathering data for entry into any database of subjects and genotypic information, including typical databases derived from diseased populations. What is demonstrated herein, is the finding that the results achieved are statistically the same as the results that would be achieved if each sample is analyzed separately. Analysis of pooled samples by a method, such as the mass spectrometric methods provided herein, permits resolution of such data and quantitation of the results. [0199]
  • For factor VII the R53Q acid polymorphism was assessed. In FIG. 9, the “individual” data represent allelic frequency observed in 92 individuals reactions. The pooled data represent the allelic frequency of the same 92 individuals pooled into a single probe reaction. The concentration of DNA in the samples of individual donors is 250 nanograms. The total concentration of DNA in the pooled samples is also 250 nanograms, where the concentration of any individual DNA is 2.7 nanograms. [0200]
  • It also was shown that it is possible to reduce the DNA concentration of individuals in a pooled samples from 2.7 nanograms to 0.27 nanograms without any change in the quality of the spectrum or the ability to quantitate the amount of sample detected. Hence low concentrations of sample can be used in the pooling methods. [0201]
  • Use of the Databases and Markers Identified Thereby [0202]
  • The successful use of genomics requires a scientific hypothesis (i.e., common genetic variation, such as a SNP), a study design (i.e., complex disorders), samples and technology, such as the chip-based mass spectrometric analyses (see, e.g., U.S. Pat. No. 5,605,798, U.S. Pat. No. 5,777,324, U.S. Pat. No. 6,043,031, allowed copending U.S. application Ser. No. 08/744,481, U.S. application Ser. No. 08/990,851, International PCT application No. WO 98/20019, copending U.S. application Ser. No. 09/285,481, which describes an automated process line for analyses; see, also, U.S. application Ser. Nos. 08/617,256, 09/287,681, 09/287,682, 09/287,141 and 09/287,679, allowed copending U.S. application Ser. No. 08/744,481, International PCT application No. PCT/US97/20444, published as International PCT application No. WO 98/20019, and based upon U.S. application Ser. Nos. 08/744,481, 08/744,590, 08/746,036, 08/746,055, 08/786,988, 08/787,639, 08/933,792, 08/746,055, 09/266,409, 08/786,988 and 08/787,639; see, also U.S. application Ser. No. 09/074,936). All of these aspects can be used in conjunction with the databases provided herein and samples in the collection. [0203]
  • The databases and markers identified thereby can be used, for example, for identification of previously unidentified or unknown genetic markers and to identify new uses for known markers. As markers are identified, these can be entered into the database to use as sorting parameters from which additional correlations can be determined. [0204]
  • Previously Unidentified or Unknown Genetic Markers [0205]
  • The samples in the healthy databases can be used to identify new polymorphisms and genetic markers, using any mapping, sequencing, amplification and other methodologies, and in looking for polymorphisms among the population in the database. The thus-identified polymorphism can then be entered into the database for each sample, and the database sorted (stratified) using that polymorphism as a sorting parameter to identify any patterns and correlations that emerge, such as age correlated changes in the frequency of the identified marker. If a correlation is identified, the locus of the marker can be mapped and its function or effect assessed or deduced. [0206]
  • Thus, the databases here provide means for: [0207]
  • identification of significantly different allelic frequencies of genetic factors by comparing the occurrence or disappearance of the markers with increasing age in population and then associating the markers with a disease or a biochemical pathway; [0208]
  • identification of significantly different allelic frequencies of disease causing genetic factors by comparing the male with the female population or comparing other selected stratified populations and associating the markers with a disease or a biochemical pathway; [0209]
  • identification of significantly different allelic frequencies of disease causing genetic factors by comparing different ethnic groups and associating the markers with a disease or a biochemical pathway that is known to occur in high frequency in the ethnic group; [0210]
  • profiling potentially functional variants of genes through the general panmixed population stratified according to age, sex, and ethnic origin and thereby demonstrating the contribution of the variant genes to the physical condition of the investigated population; [0211]
  • identification of functionally relevant gene variants by gene disequilibrium analysis performed within the general panmixed population stratified according to age, sex, and ethnic origin and thereby demonstrating their contribution to the physical condition of investigated population; [0212]
  • identification of potentially functional variants of chromosomes or parts of chromosomes by linkage disequilibrium analysis performed within the general panmixed population stratified according to age, sex, and ethnic origin and thereby demonstrating their contribution to the physical condition of investigated population. [0213]
  • Uses of the Identified Markers and Known Markers [0214]
  • The databases can also be used in conjunction with known markers and sorted to identify any correlations. For example, the databases can be used for: [0215]
  • determination and evaluation of the penetrance of medically relevant polymorphic markers; [0216]
  • determination and evaluation of the diagnostic specificity of medically relevant genetic factors; [0217]
  • determination and evaluation of the positive predictive value of medically relevant genetic factors; [0218]
  • determination and evaluation of the onset of complex diseases, such as, but are not limited to, diabetes, hypertension, autoimmune diseases, arteriosclerosis, cancer and other diseases within the general population with respect to their causative genetic factors; [0219]
  • delineation of the appropriate strategies for preventive disease treatment; [0220]
  • delineation of appropriate timelines for primary disease intervention; [0221]
  • validation of medically relevant genetic factors identified in isolated populations regarding their general applicability; [0222]
  • validation of disease pathways including all potential target structures identified in isolated populations regarding their general applicability; and [0223]
  • validation of appropriate drug targets identified in isolated populations regarding their general applicability. [0224]
  • Among the diseases and disorders for which polymorphisms can be linked include, those linked to inborn errors of metabolism, acquired metabolic disorders, intermediary metabolism, oncogenesis pathways, blood clotting pathways, and DNA synthetic and repair pathways, DNA repair/replication/transcription factors and activities, e.g., such as genes related to oncogenesis, aging and genes involved in blood clotting and the related biochemical pathways that are related to thrombosis, embolism, stroke, myocardial infarction, angiogenesis and oncogenesis. [0225]
  • For example, a number of diseases are caused by or involve deficient or defective enzymes in intermediary metabolism (see, e.q., Tables 1 and 2, below) that result, upon ingestion of the enzyme substrates, in accumulation of harmful metabolites that damage organs and tissues, particularly an infant's developing brain and other organs, resulting in mental retardation and other developmental disorders. [0226]
  • Identification of Markers and Genes for Such Disorders is of Great Interest. [0227]
  • Model Systems [0228]
  • Several gene systems, p21, p53 and Lipoprotein Lipase polymorphism (N291S), were selected. The p53 gene is a tumor suppressor gene that is mutated in diverse tumor types. One common allelic variant occurs at [0229] codon 72. A polymorphism that has been identified in the p53 gene, i.e., the R72P allele, results in an amino acid exchange, arginine to proline, at codon 72 of the gene.
  • Using diseased populations, it has been shown that there are ethnic differences in the allelic distribution of these alleles among African-Americans and Caucasians in the U.S. The results here support this finding and also demonstrate that the results obtained with a healthy database are meaningful (see, FIG. 7B). [0230]
  • The 291S allele leads to reduced levels of high density lipoprotein cholesterol (HDL-C) that is associated with an increased risk of males for arteriosclerosis and in particular myocardial infarction (see, Reymer et al. (1995) [0231] Nature Genetics 10:28-34).
  • Both genetic polymorphisms were profiled within a part of the Caucasian population-based sample bank. For the polymorphism located in the lipoprotein lipase gene a total of 1025 unselected individuals (436 males and 589 females) were tested. Genomic DNA was isolated from blood samples obtained from the individuals. [0232]
  • As shown in the Examples and figures, an exemplary database containing about 5000 subjects, answers to the questionnaire (see FIG. 3), and genotypic information has been stratified. A particular known allele has been selected, and the samples tested for the marker using mass spectrometric analyses, particularly PROBE (see the EXAMPLES) to identify polymorphisms in each sample. The population in the database has been sorted according to various parameters and correlations have been observed. For example, FIGS. [0233] 2A-C, show sorting of the data by age and sex for the Lipoprotein Lipase gene in the Caucasian population in the database. The results show a decrease in the frequency of the allele with age in males but no such decrease in females. Other alleles that have been tested against the database, include, alleles of p53, p21 and factor VII. Results when sorted by age are shown in the figures.
  • These examples demonstrate an effect of altered frequency of disease causing genetic factors within the general population. The scientific interpretation of those results allows prediction of medical relevance of polymorphic genetic alterations. In addition, conclusions can be drawn with regard to their penetrance, diagnostic specificity, positive predictive value, onset of disease, most appropriate onset of preventive strategies, and the general applicability of genetic alterations identified in isolated populations to panmixed populations. [0234]
  • Therefore, an age- and sex-stratified population-based sample bank that is ethnically homogenous is a suitable tool for rapid identification and validation of genetic factors regarding their potential medical utility. [0235]
  • Exemplary Computer System for Creating, Storing and Processing the Databases [0236]
  • Systems [0237]
  • Systems, including computers, containing the databases are provided herein. The computers and databases can be used in conjunction, for example, with the APL system (see, copending U.S. application Ser. No. 09/285,481), which is an automated system for analyzing biopolymers, particularly nucleic acids. Results from the APL system can be entered into the database. [0238]
  • Any suitable computer system can be used. The computer system can be integrated into systems for sample analysis, such as the automated process line described herein (see, e.g., copending U.S. application Ser. No. 09/285,481). [0239]
  • FIG. 17 is a block diagram of a computer constructed to provide and process the databases described herein. The processing that maintains the database and performs the methods and procedures can be performed on multiple computers all having a similar construction, or can be performed by a single, integrated computer. For example, the computer through which data are added to the database can be separate from the computer through which the database is sorted, or can be integrated with it. In either arrangement, the computers performing the processing can have a construction as illustrated in FIG. 17. [0240]
  • FIG. 17 is a block diagram of an [0241] exemplary computer 1700 that maintains the database described above and performs the methods and procedures. Each computer 1700 operates under control of a central processor unit (CPU) 1702, such as a “Pentium” microprocessor and associated integrated circuit chips, available from Intel Corporation of Santa Clara, Calif., USA. A computer user can input commands and data from a keyboard and display mouse 1704 and can view inputs and computer output at a display 1706. The display is typically a video monitor or flat panel display device. The computer 1700 also includes a direct access storage device (DASD) 1707, such as a fixed hard disk drive. The memory 1708 typically comprises volatile semiconductor random access memory (RAM). Each computer can include a program product reader 1710 that accepts a program product storage device 1712, from which the program product reader can read data (and to which it can optionally write data). The program product reader can comprise, for example, a disk drive, and the program product storage device can comprise removable storage media such as a magnetic floppy disk, an optical CD-ROM disc, a CD-R disc, a CD-RW disc, or a DVD data disc. If desired, the computers can be connected so they can communicate with each other, and with other connected computers, over a network 1713. Each computer 1700 can communicate with the other connected computers over the network 1713 through a network interface 1714 that enables communication over a connection 1716 between the network and the computer.
  • The [0242] computer 1700 operates under control of programming steps that are temporarily stored in the memory 1708 in accordance with conventional computer construction. When the programming steps are executed by the CPU 1702, the pertinent system components perform their respective functions. Thus, the programming steps implement the functionality of the system as described above. The programming steps can be received from the DASD 1707, through the program product reader 1712, or through the network connection 1716. The storage drive 1710 can receive a program product, read programming steps recorded thereon and transfer the programming steps into the memory 1708 for execution by the CPU 1702. As noted above, the program product storage device 1710 can comprise any one of multiple removable media having recorded computer-readable instructions, including magnetic floppy disks and CD-ROM storage discs. Other suitable program product storage devices can include magnetic tape and semiconductor memory chips. In this way, the processing steps necessary for operation can be embodied on a program product.
  • Alternatively, the program steps can be received into the [0243] operating memory 1708 over the network 1713. In the network method, the computer receives data including program steps into the memory 1708 through the network interface 1714 after network communication has been established over the network connection 1716 by well-known methods that will be understood by those skilled in the art without further explanation. The program steps are then executed by the CPU 1702 to implement the processing of the Garment Database system.
  • It should be understood that all of the computers of the system and can have a construction similar to that shown in FIG. 17. Details described with respect to the FIG. 17 [0244] computer 1700 will be understood to apply to all computers of the system 1700. This is indicated by multiple computers 1700 shown connected to the network 1713. Any one of the computers 1700 can have an alternative construction, so long as they can communicate with the other computers and support the functionality described herein.
  • FIG. 18 is a flow diagram that illustrates the processing steps performed using the computer illustrated in FIG. 17, to maintain and provide access to the databases, such as for identifying polymorphic genetic markers. In particular, the information contained in the database is stored in computers having a construction similar to that illustrated in FIG. 17. The first step for maintaining the database, as indicated in FIG. 18, is to identify healthy members of a population. As noted above, the population members are subjects that are selected only on the basis of being healthy, and where the subjects are mammals, such as humans, they can be selected based upon apparent health and the absence of detectable infections. The step of identifying is represented by the flow diagram box numbered 1802. [0245]
  • The next step, represented by the flow diagram box numbered 1804, is to obtain identifying and historical information and data relating to the identified members of the population. The information and data comprise parameters for each of the population members, such as member age, ethnicity, sex, medical history, and ultimately genotypic information. Initially, the parameter information is obtained from a questionnaire answered by each member, from whom a body tissue or body fluid sample also is obtained. The step of entering and storing these parameters into the database of the computer is represented by the flow diagram box numbered 1806. As additional information about each population member and corresponding sample is obtained, this information can be inputted into the database and can serve as a sorting parameter. [0246]
  • In the next step, represented by the flow diagram box numbered 1808, the parameters of the members are associated with an indexer. This step can be executed as part of the database storage operation, such as when a new data record is stored according to the relational database structure and is automatically linked with other records according to that structure. The [0247] step 1806 also can be executed as part of a conventional data sorting or retrieval process, in which the database entries are searched according to an input search or indexing key value to determine attributes of the data. For example, such search and sort techniques can be used to follow the occurrence of known genetic markers and then determine if there is a correlation with diseases for which they have been implicated. Examples of this use are for assessing the frequencies of the p53 and Lipoprotein Lipase polymorphisms.
  • Such searching of the database also can be valuable for identifying one or more genetic markers whose frequency changes within the population as a function of age, ethnic group, sex, or some other criteria. This can allow the identification of previously unknown polymorphisms and, ultimately, identification of a gene or pathway involved in the onset and progression of disease. [0248]
  • In addition, the database can be used for taking an identified polymorphism and ascertaining whether it changes in frequency when the data are sorted according to a selected parameter. [0249]
  • In this way, the databases and methods provided herein permit, among other things, identification of components, particularly key components, of a disease process by understanding its genetic underpinnings, and also an understanding of processes, such as individual drug responses. The databases and methods provided herein also can be used in methods involving elucidation of pathological pathways, in developing new diagnostic assays, identifying new potential drug targets, and in identifying new drug candidates. [0250]
  • Morbidity and/or Early Mortality Associated Polymorphisms [0251]
  • A database containing information provided by a population of healthy blood donors who were not selected for any particular disease to can be used to identify polymorphisms and the alleles in which they are present, whose frequency decreases with age. These can represent morbidity susceptibility markers and genes. [0252]
  • Polymorphisms of the genome can lead to altered gene function, protein function or genome instability. To identify those polymorphisms which have a clinical relevance/utility is the goal of a world-wide scientific effort. It can be expected that the discovery of such polymorphisms will have a fundamental impact on the identification and development of novel drug compounds to cure diseases. The strategy to identify valuable polymorphisms is cumbersome and dependent upon the availability of many large patient and control cohorts to show disease association. In particular, genes that cause a general risk of the population to suffer from any disease (morbidity susceptibility genes) will escape these case/control studies entirely. [0253]
  • Here described is a screening strategy to identify morbidity susceptibility genes underlying a variety of different diseases. The definition of a morbidity susceptibility gene is a gene that is expressed in many different cell types or tissues (housekeeping gene) and its altered function can facilitate the expression of a clinical phenotype caused by disease-specific susceptibility genes that are involved in a pathway specific for this disorder. In other words, these morbidity susceptibility genes predispose people to develop a distinct disease according to their genetic make-up for this disease. [0254]
  • Candidates for morbidity susceptibility genes can be found at the bottom level of pathways involving transcription, translation, heat-shock proteins, protein trafficking, DNA repair, assembly systems for subcellular structures (e.g. mitochondria, peroxysomes and other cellular microbodies), receptor signaling cascades, immunology, etc. Those pathways control the quality of life at the cellular level as well as for the entire organism. Mutations/polymorphisms located in genes encoding proteins for those pathways can reduce the fitness of cells and make the organism more susceptible to express the clinical phenotype caused by the action of a disease-specific susceptibility gene. Therefore, these morbidity susceptibility genes can be potentially involved in a whole variety of different complex diseases if not in all. Disease-specific susceptibility genes are involved in pathways that can be considered as disease-specific pathways like glucose-, lipid, hormone metabolism, etc. [0255]
  • The exemplified method permit, among other things, identification of genes and/or gene products involved in a man's general susceptibility to morbidity and/or mortality; use of these genes and/or gene products in studies to elucidate the genetic underpinnings of human diseases; use of these genes and/or gene products in combinatorial statistical analyses without or together with disease-specific susceptibility genes; use of these genes and/or gene products to predict penetrance of disease susceptibility genes; use of these genes and/or gene products in predisposition and/or acute medical diagnostics and use of these genes and/or gene products to develop drugs to cure diseases and/or to extend the life span of humans. [0256]
  • Screening Process [0257]
  • The healthy population stratified by age, gender and ethnicity, etc. is a very efficient and a universal screening tool for morbidity associated genes. Changes of allelic frequencies in the young compared to the old population are expected to indicate putative morbidity susceptibility genes. Individual samples of this healthy population base can be pooled to further increase the throughput. In an experiment, pools of young and old Caucasian females and males were applied to screen more than 400 randomly chosen single nucleotide polymorphisms located in many different genes. Candidate polymorphisms were identified if the allelic difference was greater than 8% between young and old for both or only one of the genders. The initial results were assayed again in at least one independent subsequent experiments. Repeated experiments are necessary to recognize unstable biochemical reactions, which occur with a frequency of about 2-3% and can mimic age-related allelic frequency differences. Average frequency differences and standard deviations are calculated after successful reproducibility of initial results. The final allelic frequency is then compared to a reference population of Caucasian CEPH sample pool. The result should show similar allelic frequencies in the young Caucasian population. Subsequently, the exact allele frequencies of candidates including genotype information were obtained by analyzing all individual samples. This procedure is straight forward with regard to time and cost. It enables the screening of an enormous number of SNPs. So far, several markers with a highly significant association to age were identified and described below. [0258]
  • In general at least 5 individuals in a stratified population should to be screened to produce statistically significant results. The frequency of the allele is determined for an age stratified population. Chi square analysis is then performed on the allelic frequencies to determine if the difference between age groups is statistically significant. A p value less than of 0.1 is considered to represent a statistically significant difference. Typically the p value should be less than 0.05. [0259]
  • Clinical Trials [0260]
  • The identification of markers whose frequency in a population decreases with age also allows for better designed and balanced clinical trials. Currently, if a clinical trial utilizes a marker as a significant endpoint in a study and the marker disappears with age, then the results of the study can be inaccurate. By using methods provided herein, it can be ascertained that if a marker decreases in frequency with age. This information can be considered and controlled when designing the study. For, example, an age independent marker could be substituted in its place. [0261]
  • The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. [0262]
  • EXAMPLE 1
  • This example describes the use of a database containing information provided by a population of healthy blood donors who were not selected for any particular disease to determine the distribution of allelic frequencies of known genetic markers with age and by sex in a Caucasian subpopulation of the database. The results described in this example demonstrate that a disease-related genetic marker or polymorphism can be identified by sorting a healthy database by a parameter or parameters, such as age, sex and ethnicity. [0263]
  • Generating a Database [0264]
  • Blood was obtained by venous puncture from human subjects who met blood bank criteria for donating blood. The blood samples were preserved with EDTA at pH 8.0 and labeled. Each donor provided information such as age, sex, ethnicity, medical history and family medical history. Each sample was labeled with a barcode representing identifying information. A database was generated by entering, for each donor, the subject identifier and information corresponding to that subject into the memory of a computer storage medium using commercially available software, e.g., Microsoft Access. [0265]
  • Model Genetic Markers [0266]
  • The frequencies of polymorphisms known to be associated at some level with disease were determined in a subpopulation of the subjects represented in the database. These known polymorphisms occur in the p21, p53 and Lipoprotein Lipase genes. Specifically, the N291S polymorphism (N291S) of the Lipoprotein Lipase gene, which results in a substitution of a serine for an asparagine at amino acid codon 291, leads to reduced levels of high density lipoprotein cholesterol (HDL-C) that is associated with an increased risk of males for arteriosclerosis and in particular myocardial infarction (see, Reymer et al. (1995) [0267] Nature Genetics 10:28-34).
  • The p53 gene encodes a cell cycle control protein that assesses DNA damage and acts as a transcription factor regulating genes that control cell growth, DNA repair and apoptosis (programmed cell death). Mutations in the p53 gene have been found in a wide variety of different cancers, including different types of leukemia, with varying frequency. The loss of normal p53 function results in genomic instability an uncontrolled cell growth. A polymorphism that has been identified in the p53 gene, i.e., the R72P allele, results in the substitution of a proline for an arginine at [0268] amino acid codon 72 of the gene.
  • The p21 gene encodes a cyclin-dependent kinase inhibitor associated with G1phase arrest of normal cells. Expression of the p21 gene triggers apoptosis. Polymorphisms of the p21 gene have been associated with Wilms' tumor, a pediatric kidney cancer. One polymorphism of the p21 gene, the S31R polymorphism, results in a substitution of an arginine for a serine at [0269] amino acid codon 31.
  • Database Analysis [0270]
  • Sorting of Subjects According to Specific Parameters [0271]
  • The genetic polymorphisms were profiled within segments of the Caucasian subpopulation of the sample bank. For p53 profiling, the genomic DNA isolated from blood from a total of 1277 Caucasian subjects age 18-59 years and 457 Caucasian subjects age 60-79 years was analyzed. For p21 profiling, the genomic DNA isolated from blood from a total of 910 Caucasian subjects age 18-49 years and 824 Caucasian subjects age 50-79 years was analyzed. For lipoprotein lipase gene profiling, the genomic DNA from a total of 1464 Caucasian females and 1470 Caucasian males under 60 years of age and a total of 478 Caucasian females and 560 Caucasian males over 60 years of age was analyzed. [0272]
  • Isolation and Analysis of Genomic DNA [0273]
  • Genomic DNA was isolated from blood samples obtained from the individuals. Ten milliliters of whole blood from each individual was centrifuged at 2000×g. One milliliter of the buffy coat was added to 9 ml of 155 mM NH[0274] 4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA, incubated 10 min at room temperature and centrifuged for 10 min at 2000×g. The supernatant was removed, and the white cell pellet was washed in 155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM Na2EDTA and resuspended in 4.5 ml of 50 mM Tris, 5 mM EDTA and 1% SDS. Proteins were precipitated from the cell lysate by 6 mM ammonium acetate, pH 7.3, and then separated from the nucleic acids by centrifugation at 3000×g. The nucleic acid was recovered from the supernatant by the addition of an equal volume of 100% isopropanol and centrifugation at 2000×g. The dried nucleic acid pellet was hydrated in 10 mM Tris, pH 7.6, and 1 mM Na2EDTA and stored at 4° C.
  • Assays of the genomic DNA to determine the presence or absence of the known genetic markers were developed using the BiomassPROBE™ detection method (primer oligo base extension) reaction. This method uses a single detection primer followed by an oligonucleotide extension step to give products, which can be readily resolved by mass spectrometry, and, in particular, MALDI-TOF mass spectrometry. The products differ in length depending on the presence or absence of a polymorphism. In this method, a detection primer anneals adjacent to the site of a variable nucleotide or sequence of nucleotides, and the primer is extended using a DNA polymerase in the presence of one or more dideoxyNTPs and, optionally, one or more deoxyNTPs. The resulting products are resolved by MALDI-TOF mass spectrometry. The mass of the products as measured by MALDI-TOF mass spectrometry makes possible the determination of the nucleotide(s) present at the variable site. [0275]
  • First, each of the Caucasian genomic DNA samples was subjected to nucleic acid amplification using primers corresponding to [0276] sites 5′ and 3′ of the polymorphic sites of the p21 (S31R allele), p53 (R72P allele) and Lipoprotein Lipase (N291S allele) genes. One primer in each primer pair was biotinylated to permit immobilization of the amplification product to a solid support. Specifically, the polymerase chain reaction primers used for amplification of the relevant segments of the p21, p53 and lipoprotein lipase genes are shown below: US4p21c31-2F (SEQ ID NO: 9) and US5p21-2R (SEQ ID NO: 10) for p21 gene amplification; US4-p53-ex4-F (also shown as p53-ex4US4 (SEQ ID NO: 2)) and US5-p53/2-4R (also shown as US5P53/4R (SEQ ID NO: 3)) for p53 gene amplification; and US4-LPL-F2 (SEQ ID NO: 16) and US5-LPL-R2 (SEQ ID NO: 17) for lipoprotein lipase gene amplification.
  • Amplification of the respective DNA sequences was conducted according to standard protocols. For example, primers can be used in a concentration of 8 pmol. The reaction mixture (e.g., [0277] total volume 50 μl) can contain Taq-polymerase including 10×buffer and dTNPs. Cycling conditions for polymerase chain reaction amplification can typically be initially 5 min. at 95° C., followed by 1 min. at 94° C., 45 sec at 53° C., and 30 sec at 72° C. for 40 cycles with a final extension time of 5 min at 72° C. Amplification products can be purified by using Qiagen's PCR purification kit (No. 28106) according to manufacturer's instructions. The elution of the purified products from the column can be done in 50 μl TE-buffer (10 mM Tris, 1 mM EDTA, pH 7.5).
  • The purified amplification products were immobilized via a biotin-avidin linkage to streptavidin-coated beads and the double-stranded DNA was denatured. A detection primer was then annealed to the immobilized DNA using conditions such as, for example, the following: 50 μl annealing buffer (20 mM Tris, 10 mM KCl, 10 mM (NH[0278] 4)2SO4, 2 mM MgSO2, 1% Triton X-100, pH 8) at 50° C. for 10 min, followed by washing of the beads three times with 200 μl washing buffer (40 mM Tris, 1 mM EDTA, 50 mM NaCl, 0.1% Tween 20, pH 8.8) and once in 200 μl TE buffer.
  • The PROBE extension reaction was performed, for example, by using some components of the DNA sequencing kit from USB (No. 70770) and dNTPs or ddNTPs from Pharmacia. An exemplary protocol could include a total reaction volume of 45 μl, containing of 21 μl water, 6 μl Sequenase-buffer, 3 [0279] μl 10 mM DTT solution, 4.5 μp, 0.5 mM of three dNTPs, 4.5 μl, 2 mM the missing one ddNTP, 5.5 μl glycerol enzyme dilution buffer, 0.25 μl Sequenase 2.0, and 0.25 pyrophosphatase. The reaction can then by pipetted on ice and incubated for 15 min at room temperature and for 5 min at 37° C. The beads can be washed three times with 200 μl washing buffer and once with 60 μl of a 70 mM NH4-Citrate solution.
  • The DNA was denatured to release the extended primers from the immobilized template. Each of the resulting extension products was separately analyzed by MALDI-TOF mass spectrometry using 3-hydroxypicolinic acid (3-HPA) as matrix and a UV laser. [0280]
  • Specifically, the primers used in the PROBE reactions are as shown below: P21/31-3 (SEQ ID NO: 12) for PROBE analysis of the p21 polymorphic site; P53/72 (SEQ ID NO: 4) for PROBE analysis of the p53 polymorphic site; and LPL-2 for PROBE analysis of the lipoprotein lipase gene polymorphic site. In the PROBE analysis of the p21 polymorphic site, the extension reaction was performed using dideoxy-C. The products resulting from the reaction conducted on a “wild-type” allele template (wherein [0281] codon 31 encodes a serine) and from the reaction conducted on a polymorphic S31R allele template (wherein codon 31 encodes an arginine) are shown below and designated as P21/31-3 Ser (wt) (SEQ ID NO: 13) and P21/31-3 Arg (SEQ ID NO: 14), respectively. The masses for each product as can be measured by MALDI-TOF mass spectrometry are also provided (i.e., 4900.2 Da for the wild-type product and 5213.4 Da for the polymorphic product).
  • In the PROBE analysis of the p53 polymorphic site, the extension reaction was performed using dideoxy-C. The products resulting from the reaction conducted on a “wild-type” allele template (wherein [0282] codon 72 encodes an arginine) and from the reaction conducted on a polymorphic R72P allele template (wherein codon 72 encodes a proline) are shown below and designated as Cod72 G Arg (wt) and Cod72 C Pro, respectively. The masses for each product as can be measured by MALDI-TOF mass spectrometry are also provided (i.e., 5734.8 Da for the wild-type product and 5405.6 Da for the polymorphic product).
  • In the PROBE analysis of the lipoprotein lipase gene polymorphic site, the extension reaction was performed using a mixture of ddA and ddT. The products resulting from the reaction conducted on a “wild-type” allele template (wherein codon 291 encodes an asparagine) and from the reaction conducted on a polymorphic N291S allele template (wherein codon 291 encodes a serine) are shown below and designated as 291Asn and 291Ser, respectively. The masses for each product as can be measured by MALDI-TOF mass spectrometry are also provided (i.e., 6438.2 Da for the wild-type product and 6758.4 Da for the polymorphic product). [0283]
  • P53-1 (R72P) [0284]
    PCR Product length: 407 bp
                                            US4-p53-ex4-F
                                            ctg aggacctggt cctctgactg (SEQ ID NO: 1)
    ctcttttcac ccatctacag tcccccttgc cgtcccaagc aatggatgat ttgatgctgt
    ccccggacga tattgaacaa tggttcactg aagacccagg tccagatgaa gctcccagaa
     P53/72              72R
    tgccagaggc tgctccccgc gtggcccctg caccagcagc tcctacaccg gcggcccctg
                       c 72P
    caccagcccc ctcctggccc ctgtcatctt ctgtcccttc ccagaaaacc taccagggca
    gctacggttt ccgtctgggc ttcttgcatt ctgggacagc caagtctgtg acttgcacgg
    tcagttgccc tgaggggctg gcttccatga gacttcaa
                                 US5-p53/2-4R
    Primers (SEQ ID NOs: 2-4)
    p53-ex4FUS4 ccc agt cac gac gtt gta aaa cgc tga gga cct ggt cct ctg ac
    US5P53/4R agc gga taa caa ttt cac aca ggt tga agt ctc atg gaa gcc
    P53/72 gcc aga ggc tgc tcc cc
  • [0285]
    Masses
    Product Termination:
    Allele ddC SEQ # Length Mass
    P53/72 gccagaggctgctcccc 5 17 5132.4
    Cod72 G Arg gccagaggctgctccccgc 6 19 5734.8
    (wt)
    Cod72 C Pro gccagaggctgctccccc 7 18 5405.6
  • Biotinylated US5 primer is used in the PCR amplification. [0286]
  • LPL-1 (N291S) [0287]
  • Amino acid exchange asparagine to serine at codon 291 of the lipoprotein lipase gene. [0288]
    PCR Product length: 251 bp
    US4-LPL-F2 (SEQ ID NO: 16)
    gcgctccatt catctcttca tcgactctct gttgaatgaa gaaaatccaa gtaaggccta (SEQ ID NO: 15)
    caggtgcagt tccaaggaag cctttgagaa agggctctgc ttgagttgta gaaagaaccg
                LPL-2             291N
    ctgcaacaat ctgggctatg agatcaataa agtcagagcc aaaagaagca gcaaaatgta
                                g 291S
    cctgaagact cgttctcaga tgccc
                    US4-LPL-R2
    Primers (SEQ ID NOs: 16-18):
    US4-LPL-F2 ccc agt cac gac gtt gta aaa cgg cgc tcc att cat ctc ttc
    US5-LPL-R2 agc gga taa caa ttt cac aca ggg ggc atc tga gaa cga gtc
    LPL-2 caa tct ggg cta tga gat ca
  • [0289]
    Masses
    Allele Product Termination: ddA, ddT SEQ # Length Mass
    LPL-2 caatctgggctatgagatca 19 20 6141
    291 Asn caatctgggctatgagatcaa 20 21 6438.2
    291 Ser caatctgggctatgagatcagt 21 22 6758.4
  • Biotinylated US5 primer is used in the PCR amplification. [0290]
    P21-1 (S31R)
    Amino acid exchange serine to arginine at codon 31 of the tumor
    suppressor gene p21. Product length: 207 bp
    US4p21c3l-2F
                                             gtcc gtcagaaccc atgcggcagc (SEQ ID NO: 8)
                                           p21/31-3 31S
    aaggcctgcc gccgcctctt cggcccagtg gacagcgagc agctgagccg cgactgtgat
                                                       a 31R
    gcgctaatgg cgggctgcat ccaggaggcc cgtgagcgat ggaacttcga ctttgtcacc
    gagacaccac tggaggg
             US5p21-2R
    Primers (SEQ ID NOs: 9-11)
    US4p21c31-2F ccc agt cac gac gtt gta aaa cgg tcc gtc aga acc cat gcg g
    US5p21-2R agc gga taa caa ttt cac aca ggc tcc agt ggt gtc tcg gtg ac
    P21/31-3 cag cga gca gct gag
  • [0291]
    Masses
    Allele Product Termination: ddC SEQ # Length Mass
    P21/31-3 cagcgagcagctgag 12 15 4627
    P21/31-3 Ser cagcgagcagctgagc 13 16 4900.2
    (wt)
    P21/31-3 Arg cagcgagcagctgagac 14 17 5213.4
  • Biotinylated US5 primer is used in the PCR amplification. [0292]
  • Each of the Caucasian subject DNA samples was individually analyzed by MALDI-TOF mass spectrometry to determine the identity of the nucleotide at the polymorphic sites. The genotypic results of each assay can be entered into the database. The results were then sorted according to age and/or sex to determine the distribution of allelic frequencies by age and/or sex. As depicted in the Figures showing histograms of the results, in each case, there was a differential distribution of the allelic frequencies of the genetic markers for the p21, p53 and lipoprotein lipase gene polymorphisms. [0293]
  • FIG. 8 shows the results of the p21 genetic marker assays and reveals a statistically significant decrease (from 13.3% to 9.2%) in the frequency of the heterozygous genotype (S31 R) in Caucasians with age (18-49 years of age compared to 50-79 years of age). The frequencies of the homozygous (S31 and R31) genotypes for the two age groups are also shown, as are the overall frequencies of the S31 and R31 alleles in the two age groups (designated as *S31 and *R31, respectively in the Figure). [0294]
  • FIGS. [0295] 7A-C show the results of the p53 genetic marker assays and reveals a statistically significant decrease (from 6.7% to 3.7%) in the frequency of the homozygous polymorphic genotype (P72) in Caucasians with age (18-59 years of age compared to 60-79 years of age). The frequencies of the homozygous “wild-type” genotype (R72) and the heterozygous genotype (R72P) for the two age groups are also shown, as are the overall frequencies of the R72 and P72 alleles in the two age groups (designated as *R72 and *P72, respectively in the Figure). These results are consistent with the observation that allele is not benign, as p53 regulates expression of a second protein, p21, which inhibits cyclin-dependent kinases (CDKs) needed to drive cells through the cell-cycle (a mutation in either gene can disrupt the cell cycle leading to increased cell division).
  • FIG. 2C shows the results of the lipoprotein lipase gene genetic marker assays and reveals a statistically significant decrease (from 1.97% to 0.54%) in the frequency of the polymorphic allele (S291) in Caucasian males with age (see also Reymer et al. (1995) [0296] Nature Genetics 10:28-34). The frequencies of this allele in Caucasian females of different age groups are also shown.
  • EXAMPLE 2
  • This example describes the use of MALDI-TOF mass spectrometry to analyze DNA samples of a number of subjects as individual samples and as pooled samples of multiple subjects to assess the presence or absence of a polymorphic allele (the 353Q allele) of the Factor VII gene and determine the frequency of the allele in the group of subjects. The results of this study show that essentially the same allelic frequency can be obtained by analyzing pooled DNA samples as by analyzing each sample separately and thereby demonstrate the quantitative nature of MALDI-TOF mass spectrometry in the analysis of nucleic acids. [0297]
  • Factor VII [0298]
  • Factor VII is a serine protease involved in the extrinsic blood coagulation cascade. This factor is activated by thrombin and works with tissue factor (Factor III) in the processing of Factor X to Factor Xa. There is evidence that supports an association between polymorphisms in the Factor VII gene and increased Factor VII activity which can result in an elevated risk of ischemic cardiovascular disease, including myocardial infarction. The polymorphism investigated in this study is R353Q (i.e., a substitution of a glutamic acid residue for an arginine residue at codon 353 of the Factor VII gene) (see Table 5). [0299]
  • Analysis of DNA Samples for the Presence or Absence of the 353Q Allele of the Factor VII Gene [0300]
  • Genomic DNA was isolated from separate blood samples obtained from a large number of subjects divided into multiple groups of 92 subjects per group. Each sample of genomic DNA was analyzed using the BiomassPROBE™ assay as described in Example 1 to determine the presence or absence of the 353Q polymorphism of the Factor VII gene. [0301]
  • First, DNA from each sample was amplified in a polymerase chain reaction using primers F7-353FUS4 (SEQ ID NO: 24) and F7-353RUS5 (SEQ ID NO: 26) as shown below and using standard conditions, for example, as described in Example 1. One of the primers was biotinylated to permit immobilization of the amplification product to a solid support. The purified amplification products were immobilized via a biotin-avidin linkage to streptavidin-coated beads and the double-stranded DNA was denatured. A detection primer was then annealed to the immobilized DNA using conditions such as, for example, described in Example 1. The detection primer is shown as F7-353-P (SEQ ID NO: 27) below. The PROBE extension reaction was carried out using conditions, for example, such as those described in Example 1. The reaction was performed using ddG. [0302]
  • The DNA was denatured to release the extended primers from the immobilized template. Each of the resulting extension products was separately analyzed by MALDI-TOF mass spectrometry. A matrix such as 3-hydroxypicolinic acid (3-HPA) and a UV laser could be used in the MALDI-TOF mass spectrometric analysis. The products resulting from the reaction conducted on a “wild-type” allele template (wherein codon 353 encodes an arginine) and from the reaction conducted on a polymorphic 353Q allele template (wherein codon 353 encodes a glutamic acid) are shown below and designated as 353 CGG and 353 CAG, respectively. The masses for each product as can be measured by MALDI-TOF mass spectrometry are also provided (i.e., 5646.8 Da for the wild-type product and 5960 Da for the polymorphic product). [0303]
  • The MALDI-TOF mass spectrometric analyses of the PROBE reactions of each DNA sample were first conducted separately on each sample (250 nanograms total concentration of DNA per analysis). The allelic frequency of the 353Q polymorphism in the group of 92 subjects was calculated based on the number of individual subjects in which it was detected. [0304]
  • Next, the samples from 92 subjects were pooled (250 nanograms total concentration of DNA in which the concentration of any individual DNA is 2.7 nanograms), and the pool of DNA was subjected to MALDI-TOF mass spectrometric analysis. The area under the signal corresponding to the mass of the 353Q polymorphism PROBE extension product in the resulting spectrum was integrated in order to quantitate the amount of DNA present. The ratio of this amount to total DNA was used to determine the allelic frequency of the 353Q polymorphism in the group of subjects. This type of individual sample vs. pooled sample analysis was repeated for numerous different groups of 92 different samples. [0305]
  • The frequencies calculated based on individual MALDI-TOF mass spectrometric analysis of the 92 separate samples of each group of 92 are compared to those calculated based on MALDI-TOF mass spectrometric analysis of pools of DNA from 92 samples in FIG. 9. These comparisons are shown as “pairs” of bar graphs in the Figure, each pair being labeled as a separate “pool” number, e.g., P1, P16, P2, etc. Thus, for example, for P1, the allelic frequency of the polymorphism calculated by separate analysis of each of the 92 samples was 11.41%, and the frequency calculated by analysis of a pool of all of the 92 DNA samples was 12.09%. [0306]
  • The similarity in frequencies calculated by analyzing separate DNA samples individually and by pooling the DNA samples demonstrates that it is possible, through the quantitative nature of MALDI-TOF mass spectrometry, to analyze pooled samples and obtain accurate frequency determinations. The ability to analyze pooled DNA samples significantly reduces the time and costs involved in the use of the non-selected, healthy databases as described herein. It has also been shown that it is possible to decrease the DNA concentration of the individual samples in a pooled mixture from 2.7 nanograms to 0.27 nanograms without any change in the quality of the spectrum or the ability to quantitate the amount of sample detected. [0307]
  • Factor VII R353Q PROBE Assay [0308]
  • PROBE Assay for cod353 CGG>CAG (Arg>Gln), Exon 9 G>A. [0309]
    PCR fragment: 134 bp (incl. US tags; SEQ ID Nos. 22 and 23)
    Frequency of A allele: Europeans about 0.1, Japanese/Chinese about
    0.03-0.05 (Thromb. Haemost. 1995, 73:617-22; Diabetologia 1998,
    41:760-6):
                  F7-353FUS4>
    1201 GTGCCGGCTA CTCGGATGGC AGCAAGGACT CCTGCAAGGG GGACAGTGGA GGCCCACATG
         F7-353-P>      A           <F7-353RUS5
    1261 CCACCCACTA CCGGGGCACG TGGTACCTGA CGGGCATCGT CAGCTGGGGC CAGGGCTGCG
    Primers (SEQ ID NOs: 24-26) Tmgs
    F7-353FUS4 CCC AGT CAC GAC GTT GTA AAA CGA TGG CAG CAA GGA CTC CTG 64° C.
    F7-353-P CAC ATG CCA CCC ACT ACC
    F7-353RUS5 AGC GGA TAA CAA TTT CAC ACA GGT GAC GAT GCC CGT CAG GTA C 64° C.
  • [0310]
    Masses
    Allele Product Termination: ddG SEQ # Length Mass
    F7-353-P atgccacccactacc 27 18 5333.6
    353 CGG cacatgccacccactaccg 28 19 5646.8
    353 CAG cacatgccacccactaccag 29 20 5960
    US5-bio bio- agcggataacaatttcacacagg 30 23 7648.6
  • Conclusion [0311]
  • The above examples demonstrate an effect of altered frequency of disease causing genetic factors within the general population. Interpretation of those results allows prediction of the medical relevance of polymorphic genetic alterations. In addition, conclusions can be drawn with regard to their penetrance, diagnostic specificity, positive predictive value, onset of disease, most appropriate onset of preventive strategies, and the general applicability of genetic alterations identified in isolated populations to panmixed populations. Therefore, an age- and sex-stratified population-based sample bank that is ethnically homogenous is a suitable tool for rapid identification and validation of genetic factors regarding their potential medical utility. [0312]
  • EXAMPLE 3
  • Morbidity and Mortality Markers [0313]
  • Sample Band and Initial Screening [0314]
  • Healthy samples were obtained through the blood bank of San Bernardino, Calif. Donors signed prior to the blood collection a consent form and agreed that their blood will be used in genetic studies with regard to human aging. All samples were anomymized. Tracking back of samples is not possible. [0315]
  • Isolation of DNA from Blood Samples of a Healthy Donor Population [0316]
  • Blood is obtained from a donor by venous puncture and preserved with 1 mM EDTA pH 8.0. Ten milliliters of whole blood from each donor was centrifuged at 2000×g. One milliliter of the buffy coat was added to 9 milliters of 155 mM NH[0317] 4Cl, 1 OmM KHCO3, and 0.1 mM Na2EDTA, incubated 10 minutes at room temperature and centrifuged for 10 minutes at 2000×g. The supernatant was removed, and the white cell pellet was washed in 155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA and resuspended in 4.5 milliliters of 50 mM Tris, 5 mM EDTA, and 1% SDS. Proteins were precipitated from the cell lysate by 6M Ammonium Acetate, pH 7.3, and separated from the nucleic acid by centrifugation 3000×g. The nucleic acid was recovered from the supernatant by the addition of an equal volume of 100% isopropanol and centrifugation at 2000×g. The dried nucleic acid pellet was hydrated in lOmM Tris pH 7.6 and 1 mM Na2EDTA and stored at 4C.
  • In this study, samples were pooled as shown in Table 1. Both parents of the blood donors were of Caucasian origin. [0318]
    TABLE 1
    Pool ID Sex Age-range # individuals
    SP1 Female 18-39 years 276
    SP2 Males 18-39 years 276
    SP3 Females 60-69 years 184
    SP4 Males 60-79 years 368
  • More than 400 SNPs were tested using all four pools. After one test run 34 assays were selected to be re-assayed at least once. Finally, 10 assays showed repeatedly differences in allele frequencies of several percent and, therefore, fulfilled the criteria to be tested using the individual samples. Average allele frequency and standard deviation is tabulated in Table 2. [0319]
    TABLE 2
    Assay ID SP1 SP1-STD SP2 SP2-STD SP3 SP3-STD SP4 SP4-STD
    47861 0.457 0.028 0.433 0.042 0.384 0.034 0.380 0.015
    47751 0.276 0.007 0.403 0.006 0.428 0.052 0.400 0.097
    48319 0.676 0.013 0.627 0.018 0.755 0.009 0.686 0.034
    48070 0.581 0.034 0.617 0.045 0.561 n.a. 0.539 0.032
    49807 0.504 0.034 0.422 0.020 0.477 0.030 0.556 0.005
    49534 0.537 0.017 0.503 n.a. 0.623 0.023 0.535 0.009
    49733 0.560 0.006 0.527 0.059 0.546 0.032 0.436 0.016
    49947 0.754 0.008 0.763 0.047 0.736 0.052 0.689 0.025
    50128 0.401 0.022 0.363 0.001 0.294 0.059 0.345 0.013
    63306 0.697 0.012 0.674 0.013 0.712 0.017 0.719 0.005
  • So far, 7 out of the 10 potential morbidity markers were fully analyzed. Additional information about genes in which these SNPs are located was gathered through publicly available databases, including Genbank. [0320]
  • AKAPS [0321]
  • Candidate morbidity and mortality markers include housekeeping genes, such as genes involved in signal transduction. Among such genes are the A-kinase anchoring proteins (AKAPs) genes, which participate in signal transduction pathways involving protein phosphorylation. Protein phosphorylation is an important mechanism for enzyme regulation and the transduction of extracellular signals across the cell membrane in eukaryotic cells. A wide variety of cellular substrates, including enzymes, membrane receptors, ion channels and transcription factors, can be phosphorylated in response to extracellular signals that interact with cells. A key enzyme in the phosphorylation of cellular proteins in response to hormones and neurotransmitters is cyclic AMP (cAMP)-dependent protein kinase (PKA). Upon activation by cAMP, PKA thus mediates a variety of cellular responses to such extracellular signals. An array of PKA isozymes are expressed in mammalian cells. The PKAs usually exist as inactive tetramers containing a regulatory (R) subunit dimer and two catalytic (C) subunits. Genes encoding three C subunits (Cα, Cβ and Cy) and four R subunits (RIα, RIβ, RIIα and RIIβ) have been identified [see Takio et al. (1982) [0322] Proc. Natl. Acad. Sci. U.S. A. 79:2544-2548; Lee et al. (1983) Proc. Natl. Acad. Sci. U.S. A. 80:3608-3612; Jahnsen et al. (1996) J. Biol. Chem. 261:12352-12361; Clegg et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:3703-3707; and Scott (1991) Pharmacol. Ther. 50:123-145]. The type I (RI) α and type II (RII) α subunits are distributed ubiquitously, whereas RIβ and RIIβ are present mainly in brain [see. e.g., Miki and Eddy (1999) J. Biol. Chem. 274:29057-29062]. The type I PKA holoenzyme (RIα and RIβ) is predominantly cytoplasmic, whereas the majority of type II PKA (RIIα and RIIβ) associates with cellular structures and organelles [Scott (1991) Pharmacol. Ther. 50:123-1451. Many hormones and other signals act through receptors to generate cAMP which binds to the R subunits of PKA and releases and activates the C subunits to phosphorylate proteins. Because protein kinases and their substrates are widely distributed throughout cells, there are mechanisms in place in cells to localize protein kinase-mediated responses to different signals. One such mechanism involves subcellular targeting of PKAs through association with anchoring proteins, referred to as A-kinase anchoring proteins (AKAPs), that place PKAs in close proximity to specific organelles or cytoskelet al components and particular substrates thereby providing for more specific PKA interactions and localized responses [see, e.g., Scott et al. (1990) J. Biol. Chem. 265:21561-21566; Bregman et al. (1991) J. Biol. Chem. 266:7207-7213; and Miki and Eddy (1999) J. Biol. Chem. 274:29057-290621. Anchoring not only places the kinase close to the substrates, but also positions the PKA holoenzyme at sites where it can optimally respond to fluctuations in the second messenger cAMP [Mochly-Rosen (1995) Science 268:247-251; Faux and Scott (1996) Trends Biochem. Sci. 21:312-315; Hubbard and Cohen (1993) Trends Biochem. Sci. 18:172-177].
  • Up to 75% of type II PKA is localized to various intracellular sites through association of the regulatory subunit (RII) with AKAPs [see, e.g., Hausken et al. (1996) [0323] J. Biol. Chem. 271:29016-290221. RII subunits of PKA bind to AKAPs with nanomolar affinity [Carr et al. (1992) J. Biol. Chem. 267:13376-13382], and many AKAP-RII complexes have been isolated from cell extracts. RI subunits of PKA bind to AKAPs with only micromolar affinity [Burton et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:11067-110721. Evidence of binding of a PKA RI subunit to an AKAP has been reported [Miki and Eddy (1998) J. Biol. Chem 273:34384-34390] in which RIα-specific and RIα/RIIα dual specificity PKA anchoring domains were identified on FSC1/AKAP82. Additional dual specific AKAPs, referred to as D-AKAP1 and D-AKAP2, which interact with the type I and type II regulatory subunits of PKA have also been reported [Huang et al. (1997) J. Biol. Chem. 272:8057-8064; Huang et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:11184-11189].
  • More than 20 AKAPs have been reported in different tissues and species. Complementary DNAs (cDNAs) encoding AKAPs have been isolated from diverse species, ranging from [0324] Caenorhabditis elegans and Drosophilia to human [see, e.g., Colledge and Scott (1999) Trends Cell Biol. 9:216-2211. Regions within AKAPs that mediate association with RII subunits of PKA have been identified. These regions of approximately 10-18 amino acid residues vary substantially in primary sequence, but secondary structure predictions indicate that they are likely to form an amphipathic helix with hydrophobic residues aligned along one face of the helix and charged residues along the other [Carr et al. (1991) J. Biol. Chem. 266:14188-14192; Carr et al. (1992) J. Biol. Chem. 267:13376-13382]. Hydrophobic amino acids with a long aliphatic side chain, e.g., valine, leucine or isoleucine, can participate in binding to RII subunits [Glantz et al. (1993) J. Biol. Chem. 268:12796-12804].
  • Many AKAPs also have the ability to bind to multiple proteins, including other signaling enzymes. For example, AKAP79 binds to PKA, protein kinase C (PKC) and the protein phosphatase calcineurin (PP2B) [Coghlan et al. (1995) [0325] Science 267:108-112 and Klauck et al. (1996) Science 271:1589-15921. Therefore, the targeting of AKAP79 to neuronal postsynaptic membranes brings together enzymes with opposite catalytic activities in a single complex.
  • AKAPs thus serve as potential regulatory mechanisms that increase the selectivity and intensity of a cAMP-mediated response. There is a need, therefore, to identify and elucidate the structural and functional properties of AKAPs in order to gain a complete understanding of the important role these proteins μplay in the basic functioning of cells. [0326]
  • AKAP10 [0327]
  • The sequence of a human AKAP10 cDNA (also referred to as D-AKAP2) is available in the GenBank database, at accession numbers AF037439 (SEQ ID NO: 31) and NM 007202. The AKAP10 gene is located on [0328] chromosome 17.
  • The sequence of a mouse D-AKAP2 cDNA is also available in the GenBank database (see accession number AF021833). The mouse D-AKAP2 protein contains an RGS domain near the amino terminus that is characteristic of proteins that interact with Gα subunits and possess GTPase activating protein-like activity [Huang et al. (1997) [0329] Proc. Natl. Acad. Sci. U.S.A. 94:11184-11189]. The human AKAP10 protein also has sequences homologous to RGS domains. The carboxy-terminal 40 residues of the mouse D-AKAP2 protein are responsible for the interaction with the regulatory subunits of PKA. This sequence is fairly well conserved between the mouse D-AKAP2 and human AKAP10 proteins.
  • Polymorphisms of the Human AKAP10 Gene and Polymorphic AKAP10 Proteins [0330]
  • Polymorphisms of AKAP genes that alter gene expression, regulation, protein structure and/or protein function are more likely to have a significant effect on the regulation of enzyme (particularly PKA) activity, cellular transduction of signals and responses thereto and on the basic functioning of cells than polymorphisms that do not alter gene and/or protein function. Included in the polymorphic AKAPs provided herein are human AKAP10 proteins containing differing amino acid residues at position number 646. [0331]
  • Amino acid 646 of the human AKAP10 protein is located in the carboxy-terminal region of the protein within a segment that participates in the binding of R-subunits of PKAs. This segment includes the carboxy-[0332] terminal 40 amino acids.
  • The amino acid residue reported for position 646 of the human AKAP10 protein is an isoleucine. Polymorphic human AKAP10 proteins provided herein have the amino acid sequence but contain residues other than isoleucine at amino acid position 646 of the protein. In particular embodiments of the polymorphic human AKAP10 proteins provided herein, the amino acid at position 646 is a valine, leucine or phenylalanine residue. [0333]
  • An A to G Transition at Nucleotide 2073 of the Human AKAP10 Coding Sequence [0334]
  • As described herein, an allele of the human AKAP10 gene that contains a specific polymorphism at position 2073 of the coding sequence and thereby encodes a valine at position 646 has been detected in varying frequencies in DNA samples from younger and older segments of the human population. In this allele, the A at position 2073 of the AKAP10 gene coding sequence is changed from an A to a G, giving rise to an altered sequence in which the codon for amino acid 646 changes from ATT, coding for isoleucine, to GTT, coding for valine. [0335]
  • Morbidity Marker 1: Human Protein Kinase A Anchoring Protein (AKAP10-1) [0336]
  • PCR Amplification and BiomassPROBE assay detection of AKAP10-1 in a healthy donor population [0337]
  • PCR Amplification of Donor Population for [0338] AKAP 10
  • PCR primers were synthesized by OPERON using phosphoramidite chemistry. Amplification of the AKAP10 target sequence was carried out in single 50 μl PCR reaction with 100 ng-1 ug of pooled human genomic DNAs in a 50 μl PCR reaction. Individual DNA concentrations within the pooled samples were present in equal concentration with the final concentration ranging from 1-25 ng. Each reaction containing IX PCR buffer (Qiagen, Valencia, Calif.), 200 uM dNTPs, 1U Hotstar Taq polymerase (Qiagen, Valencia, Calif.), 4 mM MgCl[0339] 2, and 25 pmol of the forward primer containing the universal primer sequence and the target specific sequence 5′-TCTCAATCATGTGCATTGAGG-3′(SEQ ID NO: 45), 2 pmol of the reverse primer 5′-AGCGGATAACAATTTCACACAGGGATCACACAGCCATCAGCAG-3′ (SEQ ID NO: 46), and 10 pmol of a biotinylated universal primer complementary to the 5′ end of the PCR amplicon 5′-AGCGGATAACAATTTCACACAGG-3′(SEQ ID NO: 47). After an initial round of amplification with the target with the specific forward and reverse primer, the 5′ biotinylated universal primer then hybridized and acted as a reverse primer thereby introducing a 3′ biotin capture moiety into the molecule. The amplification protocol results in a 5′-biotinylated double stranded DNA amplicon and dramatically reduces the cost of high throughput genotyping by eliminating the need to 5′ biotin label each forward primer used in a genotyping. Thermal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (calculated temperature) with the following cycling parameters: 94° C. for 5 min; 45 cycles: 94° C. for 20 sec, 56° C. for 30 sec, 72° C. for 60 sec; 72° C. 3 min.
  • Immobilization of DNA [0340]
  • The 50 μl PCR reaction was added to 25 ul of streptavidin coated magnetic bead (Dynal) prewashed three times and resuspended in 1M NH[0341] 4Cl, 0.06M NH4OH. The PCR amplicons were allowed to bind to the beads for 15 minutes at room temperature. The beads were then collected with a magnet and the supernatant containing unbound DNA was removed. The unbound strand was released from the double stranded amplicons by incubation in 100 mM NaOH and washing of the beads three times with 10 mM Tris pH 8.0.
  • BiomassPROBE Assay Analysis of Donor Population for AKAP10-1 (clone 48319) [0342]
  • Genotyping using the BiomassPROBE assay methods was carried out by resuspending the DNA coated magnetic beads in 26 mM Tris-HCl pH 9.5, 6.5 mM MgCl[0343] 2 and 50 mM each of dTTP and 50 mM each of ddCTP, ddATP, ddGTP, 2.5U of a thermostable DNA polymerase (Ambersham) and 20 pmol of a template specific oligonucleotide PROBE primer 5′-CTGGCGCCCACGTGGTCAA-3′ (SEQ ID NO: 48) (Operon). Primer extension occurs with three cycles of oligonucleotide primer hybridization and extension. The extension products were analyzed after denaturation from the template with 50 mM NH4Cl and transfer of 150 nL each sample to a silicon chip preloaded with 150 nL of H3PA matrix material. The sample material was allowed to crystallize and was analyzed by MALDI-TOF (Bruker, PerSeptive). The SNP that is present in AKAP10-1 is a T to C transversion at nucleotide number 156277 of the sequence of a genomic clone of the AKAP10 gene (GenBank Accession No. AC005730) (SEQ ID NO: 36). SEQ ID NO: 35: represents the nucleotide sequence of human chromosome 17, which contains the genomic nucleotide sequence of the human AKAP10 gene, and SEQ ID NO: 36 represents the nucleotide sequence of human chromosome 17, which contains the genomic nucleotide sequence of the human AKAP10-1 allele. The mass of the primer used in the BioMass probe reaction was 5500.6 daltons. In the presence of the SNP, the primer is extended by the addition of ddC, which has a mass of 5773.8. The wildtype gene results in the addition of dT and ddG to the primer to produce an extension product having a mass of 6101 daltons.
  • The frequency of the SNP was measured in a population of age selected healthy individuals. Five hundred fifty-two (552) individuals between the ages of 18-39 years (276 females, 276 males) and 552 individuals between the ages of 60-79 (184 females between the ages of 60-69, 368 males between the age of 60-79) were tested for the presence of the polymorphism localized in the non-translated 3′ region of [0344] AKAP 10. Differences in the frequency of this polymorphism with increasing age groups were observed among healthy individuals. Statistical analysis showed that the significance level for differences in the allelic frequency for alleles between the “younger” and the “older” populations was p=0.0009 and for genotypes was p=0.003. Differences between age groups are significant. For the total population allele significance is p=0.0009, and genotype significance is p=0.003.
  • This marker led to the best significant result with regard to allele and genotype frequencies in the age-stratified population. FIG. 19 shows the allele and genotype frequency in both genders as well as in the entire population. For the latter, the significance for alleles was p=0.0009 and for genotypes was p=0.003. The young and old populations were in Hardy-Weinberg equilibrium. A preferential change of one particular genotype was not observed. [0345]
  • The polymorphism is localized in the non-translated 3′-region of the gene encoding the human protein kinase A anchoring protein (AKAP10). The gene is located on [0346] chromosome 17. Its structure includes 15 exons and 14 intervening sequences (introns). The encoded protein is responsible for the sub-cellular localization of the cAMP-dependent protein kinase and, therefore, plays a key role in the G-protein mediated receptor-signaling pathway (Huang et al. PNAS (1007) 94:11184-11189). Since its localization is outside the coding region, this polymorphism is most likely in linkage disequilibrium (LD) with other non-synonymous polymorphisms that could cause amino acid substitutions and subsequently alter the function of the protein. Sequence comparison of different Genbank database entries concerning this gene revealed further six potential polymorphisms of which two are supposed to change the respective amino acid (see Table 3).
    TABLE 3
    Exon Codon Nucleotides Amino acid
    3 100 GCT > GCC Ala > Ala
    4 177 AGT > GTG Met > Val
    8 424 GGG > GGC Gly > Gly
    10 524 CCG > CTG Pro > Leu
    12 591 GTG > GTC Val > Val
    12 599 CGC > CGA Arg > Arg
  • Morbitity Marker 2: Human Protein Kinase A Anchoring Protein (AKAP10-5) [0347]
  • Discovery of AKAP10-5 Allele (SEQ ID NO: 33) [0348]
  • Genomic DNA was isolated from blood (as described above) of seventeen (17) individuals with a genotype CC at the AKAP10-1 gene locus and a single heterozygous individual (CT) (as described). A target sequence in the AKAP10-1 gene which encodes the C-terminal PKA binding domain was amplified using the polymerase chain reaction. PCR primers were synthesized by OPERON using phosphoramidite chemistry. Amplification of the AKAP10-1 target sequence was carried out in individual 50 μl PCR reaction with 25 ng of human genomic DNA templates. Each reaction containing I×PCR buffer (Qiagen, Valencia, Calif.), 200 μM dNTPs, IU Hotstar Taq polymerase (Qiagen, Valencia, Calif.), 4 mM MgCl[0349] 2, 25 pmol of the forward primer (Ex13F) containing the universal primer sequence and the target specific sequence 5′-TCC CAA AGT GCT GGA ATT AC-3′ (SEQ ID NO: 53), and 2 pmol of the reverse primer (Ex14R) 5′-GTC CAA TAT ATG CAA ACA GTT G-3′ (SEQ ID NO: 54). Thermal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (MJ Research, Waltham, Mass.) (calculated temperature) with the following cycling parameters: 94° C. for 5 min; 45 cycles; 94° C. for 20 sec, 56° C. for 30 sec, 72° C. for 60 sec; 72° C. 3 min. After amplification the amplicons were purified using a chromatography (Mo Bio Laboratories (Solana Beach, Calif.)).
  • The sequence of the 18 amplicons, representing the target region, was determined using a standard Sanger cycle sequencing method with 25 nmol of the PCR amplicon, 3.2 uM [0350] DNA sequencing primer 5′-CCC ACA GCA GTT AAT CCT TC-3′(SEQ ID NO: 55), and chain terminating dRhodamine labeled 2′, 3′ dideoxynucleotides (PE Biosystems, Foster City, Calif.) using the following cycling parameters: 96° C. for 15 seconds; 25 cycles: 55° C. for 15 seconds, 60° C. for 4 minutes. The sequencing products precipitated by 0.3M NaOAc and ethanol. The precipitate was centrifuged and dried. The pellets were resuspended in deionized formamide and separated on a 5% polyacrylimide gel. The sequence was determined using the “Sequencher” software (Gene Codes, Ann Arbor, Mich.).
  • The sequence of all 17 of the amplicons, which are homozygous for the AKAP10-1 SNP of the amplicons, revealed a polymorphism at nucleotide position 152171 (numbering for GenBank Accession No. AC005730 for AKAP10 genomic clone (SEQ ID NO: 35)) with A replaced by G. This SNP also can be designated as located at nucleotide 2073 of a cDNA clone of the wildtype AKAP10 (GenBank Accession No. AF037439) (SEQ ID NO: 31). The amino acid sequence of the human AKAP10 protein is provided as SEQ ID NO: 34. This single nucleotide polymorphism was designated as AKAP10-5 (SEQ ID NO: 33) and resulted in a substitution of a valine for an isoleucine residue at amino acid position 646 of the amino acid sequence of human AKAP10 (SEQ ID NO: 32). [0351]
  • PCR Amplification and BiomassPROBE Assay Detection of AKAP10-5 in a Healthy Donor Population [0352]
  • The healthy population stratified by age is a very efficient and a universal screening tool for morbidity associated genes by allowing for the detection of changes of allelic frequencies in the young compared to the old population. Individual samples of this healthy population base can be pooled to further increase the throughput. [0353]
  • Healthy samples were obtained through the blood bank of San Bernardino, Calif. Both parents of the blood donors were of Caucasian origin. Practically a healthy subject, when human, is defined as human donor who passes blood bank criteria to donate blood for eventual use in the general population. These criteria are as follows: free of detectable viral, bacterial, mycoplasma, and parasitic infections; not anemic; and then further selected based upon a questionnaire regarding history (see FIG. 3). Thus, a healthy population represents an unbiased population of sufficient health to donate blood according to blood bank criteria, and not further selected for any disease state. Typically such individuals are not taking any medications. [0354]
  • PCR primers were synthesized by OPERON using phosphoramidite chemistry. Amplification of the AKAP10 target sequence was carried out in a single 50 μl PCR reaction with 100 ng-1 μg of pooled human genomic DNAs in a 50 μl PCR reaction. Individual DNA concentrations within the pooled samples were present in equal concentration with the final concentration ranging from 1-25 ng. Each reaction contained 1×PCR buffer (Qiagen, Valencia, Calif.), 200 μM dNTPs, 1U Hotstar Taq polymerase (Qiagen, Valencia, Calif.), 4 mM MgCl[0355] 2, and 25 pmol of the forward primer containing the universal primer sequence and the target specific sequence 5′-AGCGGATAACAATTTCACACAGGGAGCTAGCTTGGAAGAT TGC-3′ (SEQ ID NO: 41), 2 pmol of the reverse primer 5′-GTCCAATATATGCAAACAGTTG-3′ (SEQ ID NO: 54), and 10 pmol of a biotinylated universal primer complementary to the 5′ end of the PCR amplicon BIO:5′-AGCGGATAACAATTTCACACAGG-3′ (SEQ ID NO: 43). After an initial round of amplification with the target with the specific forward and reverse primer, the 5′ biotinylated universal primer can then be hybridized and acted as a forward primer thereby introducing a 5′ biotin capture moiety into the molecule. The amplification protocol resulted in a 5′-biotinylated double stranded DNA amplicon and dramatically reduced the cost of high throughput genotyping by eliminating the need to 5′ biotin label every forward primer used in a genotyping.
  • Themal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (calculated temperature) with the following cycling parameters: 94° C. for 5 min; 45 cycles: 94° C. for 20 sec, 56° C. for 30 sec; 72° C. for 60 sec; 72° C. 3 min. [0356]
  • Immobilization of DNA [0357]
  • The 50 μl PCR reaction was added to 25 μL of streptavidin coated magnetic beads (Dynal, Oslo, Norway), which were prewashed three times and resuspended in 1M NH[0358] 4Cl, 0.06M NH4OH. The 5′ end of one strand of the double stranded PCR amplicons were allowed to bind to the beads for 15 minutes at room temperature. The beads were then collected with a magnet, and the supernatant containing unbound DNA was removed. The hybridized but unbound strand was released from the double stranded amplicons by incubation in 100 mM NaOH and washing of the beads three times with 10 mM Tris pH 8.0.
  • Detection of AKAP10-5 using BiomassPROBE™ Assay [0359]
  • BiomassPROBE™ assay of primer extension analysis (see, U.S. Pat. No. 6,043,031) of donor population for AKAP 10-5 (SEQ ID NO: 33) was performed. Genotyping using these methods was carried out by resuspending the DNA coated magnetic beads in 26 mM Tris-HCL pH 9.5, 6.5 mM MgCl[0360] 2, 50 mM dTTP, 50 mM each of ddCTP, ddATP, ddGTP, 2.5U of a thermostable DNA polymerase (Ambersham), and 20 pmol of a template specific oligonucleotide PROBE primer 5′-ACTGAGCCTGCTGCATAA-3′ (SEQ ID NO: 44) (Operon). Primer extension occurs with three cycles of oligonucleotide primer with hybridization and extension. The extension products were analyzed after denaturation from the template with 50 mM NH4Cl and transfer of 150 nL of each sample to a silicon chip preloaded with 150 nl of H3PA matrix material. The sample material was allowed to crystallize and analyzed by MALDI-TOF (Bruker, PerSeptive). The primer has a mass of 5483.6 daltons. The SNP results in the addition of a ddC to the primer, giving a mass of 5756.8 daltons for the extended product. The wild type results in the addition a T and ddG to the primer giving a mass of 6101 daltons.
  • The frequency of the SNP was measured in a population of age selected healthy individuals. Seven hundred thirteen (713) individuals under 40 years of age (360 females, 353 males) and 703 individuals over 60 years of age (322 females, 381 males) were tested for the presence of the SNP, AKAP10-5 (SEQ ID NO: 33). Results are presented below in Table 4. [0361]
    TABLE 4
    AKAP10-5 (2073V) frequency comparison in 2 age groups
    <40 >60 delta G allele
    Female Alleles *G 38.6 34.6 4.0
    *A 61.4 65.4
    Genotypes G 13.9 11.8 2.1
    GA 49.4 45.7
    A 36.7 42.5
    Male Alleles *G 41.4 37.0 4.4
    *A 58.6 63.0
    Genotypes G 18.4 10.8 7.7
    GA 45.9 52.5
    A 35.7 36.7
    Total Alleles *G 40.0 35.9 4.1
    *A 60.0 64.1
    Genotypes G 16.1 11.2 4.9
    GA 47.7 49.4
    A 36.2 39.4
  • FIG. 20 graphically shows these results of allele and genotype distribution in the age and sex stratified Caucasian population. [0362]
  • Morbidity Marker 3: Human Methionine Sulfoxide Reductase A (msrA) [0363]
  • The age-related allele and genotype frequency of this marker in both genders and the entire population is shown in FIG. 21. The decrease of the homozygous CC genotype in the older male population is highly significant. [0364]
  • Methionine Sulfoxide Reductase A (#63306) [0365]
  • PCR Amplification and BiomassPROBE assay detection of the human methioine sulfoxide reductase A (h-msr-A) in a healthy donor population [0366]
  • PCR Amplification of Donor Population for h-msr-A [0367]
  • PCR primers were synthesized by OPERON using phosphoramidite chemistry. Amplification of the AKAP10 target sequence was carried out in single 50 μl PCR reaction with 100 ng-1 ug of pooled human genomic DNA templates in a 50 μl PCR reaction. Individual DNA concentrations within the pooled samples were present in an equal concentration with the final concentration ranging from 1-25 ng. Each reaction containing I X PCR buffer (Qiagen, Valencia, Calif.), 200 μM dNTPs, 1U Hotstar Taq polymerase (Qiagen, Valencia, Calif.), 4 mM MgCl[0368] 2, 25 pmol of the forward primer containing the universal primer sequence and the target specific sequence 5′-TTTCTCTGCACAGAGAGGC-3′ (SEQ ID NO: 49), 2 pmol of the reverse primer 5′-AGCGGATAACAATTTCACACAGGGCTGAAATCCTTCGCTTTACC-3′ (SEQ ID NO: 50), and 10 pmol of a biotinylated universal primer complementary to the 5′ end of the PCR amplicon 5′-AGCGGATAACAATTTCACACAGG-3′ (SEQ ID NO: 51). After an initial round of amplification of the target with the specific forward and reverse primers, the 5′ biotinylated universal primer was then hybridized and acted as a reverse primer thereby introducing a 3′ biotin capture moiety into the molecule. The amplification protocol results in a 5′-biotinylated double stranded DNA amplicon and dramatically reduces the cost of high throughput genotyping by eliminating the need to 5′ biotin label each forward primer used in a genotyping. Thermal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (calculated temperature) with the following cycling parameters: 94° C. for 5 min; 45 cycles: 94° C. for 20 sec, 56° C. for 30 sec, 72° C. for 60 sec; 72° C. 3 min.
  • Immobilization of DNA [0369]
  • The 50 μl PCR reaction was added to 25 ul of streptavidin coated magnetic bead (Dynal) prewashed three times and resuspended in 1M NH[0370] 4Cl, 0.06M NH4OH. The PCR amplicons were allowed to bind to the beads for 15 minutes at room temperature. The beads were then collected with a magnet and the supernatant containing unbound DNA was removed. The unbound strand was released from the double stranded amplicons by incubation in 100 mM NaOH and washing of the beads three times with 10 mM Tris pH 8.0.
  • BiomassPROBE Assay Analysis of Donor Population for h-msr A [0371]
  • Genotyping using the BiomassPROBE assay methods was carried out by resuspending the DNA coated magnetic beads in 26 mM Tris-HCl pH 9.5, 6.5 mM MgCl[0372] 2, 50 mM of dTTPs and 50 mM each of ddCTP, ddATP, ddGTP, 2.5U of a thermostable DNA polymerase (Ambersham), and 20 pmol of a template specific oligonucleotide PROBE primer 5′-CTGAAAAGGGAGAGAAAG-3′ (Operon) (SEQ ID NO: 52). Primer extension occurs with three cycles of oligonucleotide primer with hybridization and extension. The extension products were analyzed after denaturation from the template with 50 mM NH4Cl and transfer of 150 nl each sample to a silicon chip preloaded with 150 nl of H3PA matrix material. The sample material was allowed to crystallize and analyzed by MALDI-TOF (Bruker, PerSeptive). The SNP is represented as a T to C tranversion in the sequence of two ESTs. The wild type is represented by having a T at position 128 of GenBank Accession No. AW 195104, which represents the nucleotide sequence of an EST which is a portion of the wild type human msrA gene (SEQ ID NO: 39). The SNP is presented as a C at position 129 of GenBank Accession No. AW 874187, which represents the nucleotide sequence of an EST which is a portion of an allele of the human msrA gene (SEQ ID NO: 40).
  • In a genomic sequence the SNP is represented as an A to G transversion. The primer utilized in the BioMass probe reaction had a mass of 5654.8 daltons. In the presence of the SNP the primer is extended by the incorporation of a ddC and has a mass of 5928. In the presence of the wildtype the primer is extended by adding a dT and a DDC to produce a mass of 6232.1 daltons. [0373]
  • The frequency of the SNP was measured in a population of age selected healthy individuals. Five hundred fifty-two (552) individuals between the ages of 18-39 years (276 females, 276 males and 552 individuals between the age of 60-79 (184 females between the ages of 60-69, 368 males between the age of 60-79) were tested for the presence of the polymorphism localized in the nontranslated 3′ region of h-msr-A. [0374]
  • Genotype difference between male age group among healthy individuals is significant. For the male population allele significance is p=0.0009 and genotype significance is p=0.003. The age-related allele and genotype frequency of this marker in both genders and the entire population is shown in FIG. 21. The decrease of the homozygous CC genotype in the older male population is highly significant. [0375]
  • The polymorphism is localized in the non-translated 3′-region of the gene encoding the human methionine sulfoxide reductase (h-msrA). The exact localization is 451 base pairs downstream the stop codon (TAA). It is likely that this SNP is in linkage disequilibrium (LD) with another polymorphism more upstream in the coding or promoter region; thus, it does not directly cause morbidity. The enzyme methionine sulfoxide reductase has been proposed to exhibit multiple biological functions. It can serve to repair oxidative protein damage but also play an important role in the regulation of proteins by activation or inactivation of their biological functions (Moskovitz et al. (1990) PNAS 95:14071-14075). It has also been shown that its activity is significantly reduced in brain tissues of Alzheimer patients (Gabbita et al., (1999) J. Neurochem 73:1660-1666). It is scientifically conceivable that proteins involved in the metabolism of reactive oxygen species are associated to disease. [0376]
  • Conclusion [0377]
  • The use of the healthy population provides for the identification of morbidity markers. The identification of proteins involved in the G-protein coupled signaling transduction pathway or in the detoxification of oxidative stress can be considered as convincing results. Further confirmation and validation of other potential polymorphisms already identified in silico in the gene encoding the human protein kinase A anchoring protein could even provide stronger association to morbidity and demonstrate that this gene product is a suitable pharmaceutical or diagnostic target. [0378]
  • EXAMPLE 4
  • MALDI-TOF Mass Spectrometry Analysis [0379]
  • All of the products of the enzyme assays listed below were analyzed by MALDI-TOF mass spectrometry. A diluted matrix solution (0.15 μL) containing of 10:1 3-hydroxypicolinic acid:ammonium citrate in 1:1 water:acetonitrile diluted 2.5-fold with water was pipetted onto a SpectroChip (Sequenom, Inc.) and was allowed to crystallize. Then, 0.15 μL of sample was added. A linear PerSeptive Voyager DE mass spectrometer or Bruker Biflex MALDI-TOF mass spectrometer, operating in positive ion mode, was used for the measurements. The sample plates were kept at 18.2 kV for 400 nm after each UV laser shot (approximate 250 laser shots total), and then the target voltage was raised to 20 kV. The original spectra were digitized at 500 MHz. [0380]
  • EXAMPLE 5
  • Sample Conditioning [0381]
  • Where indicated in the examples below, the products of the enzymatic digestions were purified with ZipTips (Millipore, Bedford, Mass.). The ZipTips were pre-wetted with 10 μL 50% acetonitrile and equilibrated 4 times with 10 μl 0.1 M TEAAc. The oligonucleotide fragments were bound to the C18 in the ZipTip material by continuous aspiration and dispension of each sample into the ZipTip. Each digested oligonucleotide was conditioned by washing with 10 μL 0.1 M TEAAc, followed by 4 washing steps with 10 μL H[0382] 2O. DNA fragments were eluted from the Ziptip with 7 μL 50% acetonitrile.
  • Any method for condition the samples can be employed. Methods for conditioning, which generally is used to increase peak resolution, are well known (see, e.g., International PCT application No. WO 98/20019). [0383]
  • EXAMPLE 6
  • DNA Glycosylase-Mediated Sequence Analysis [0384]
  • DNA Glycosylases modifies DNA at each position that a specific nucleobase resides in the DNA, thereby producing abasic sites. In a subsequent reaction with another enzyme, a chemical, or heat, the phosphate backbone at each abasic site can be cleaved. [0385]
  • The glycosylase utilized in the following procedures was uracil-DNA glycosylase (UDG). Uracil bases were incorporated into DNA fragments in each position that a thymine base would normally occupy by amplifying a DNA target sequence in the presence of uracil. Each uracil substituted DNA amplicon was incubated with UDG, which cleaved each uracil base in the amplicon, and was then subjected to conditions that effected backbone cleavage at each abasic site, which produced DNA fragments. DNA fragments were subjected to MALDI-TOF mass spectrometry analysis. Genetic variability in the target DNA was then assessed by analyzing mass spectra. [0386]
  • Glycosylases specific for nucleotide analogs or modified nucleotides, as described herein, can be substituted for UDG in the following procedures. The glycosylase methods described hereafter, in conjunction with phosphate backbone cleavage and MALDI, can be used to analyze DNA fragments for the purposes of SNP scanning, bacteria typing, methylation analysis, microsatellite analysis, genotyping, and nucleotide sequencing and re-sequencing. [0387]
  • A. Genotyping [0388]
  • A glycosylase procedure was used to genotype the DNA sequence encoding UCP-2 (Uncoupling Protein 2). The sequence for UCP-2 is deposited in GenBank under accession number AF096289. The sequence variation genotyped in the following procedure was a cytosine (C-allele) to thymine (T-allele) variation at nucleotide position 4790, which results in a alanine to valine mutation at [0389] position 55 in the UCP-2 polypeptide.
  • DNA was amplified using a PCR procedure with a 50 μL reaction volume containing of 5 pmol biotinylated primer having the [0390] sequence 5′-TGCTTATCCCTGTAGCTACCCTGTCTTGGCCTTGCAGATCCAA-3′ (SEQ ID NO: 91), 15 pmol non-biotinylated primer having the sequence 5′-AGCGGATAACAATTTCACACAGGCCATCACACCGCGGTACTG-3′ (SEQ ID NO: 92), 200 μM dATP, 200 μM dCTP, 200 μM dGTP, 600 μM dUTP (to fully replace dTTP), 1.5 mM to 3 mM MgCl2, 1 U of HotStarTaq polymerase, and 25 ng of CEPH DNA. Amplification was effected with 45 cycles at an annealing temperature of 56° C.
  • The amplification product was then immobilized onto a solid support by incubating 50 μL of the amplification reaction with 5 μL of prewashed Dynabeads for 20 minutes at room temperature. The supernatant was removed, and the beads were incubated with 50 μL of 0.1 M NaOH for 5 minutes at room temperature to denature the double-stranded PCR product in such a fashion that single-stranded DNA was linked to the beads. The beads were then neutralized by three washes with 50 μL 10 mM TrisHCl (pH 8). The beads were resuspended in 10 μL of a 60 mM TrisHCl/1 mM EDTA (pH 7.9) solution, and 1 U uracil DNA glycosylase was added to the solution for 45 minutes at 37° C. to remove uracil nucleotides present in the single-stranded DNA linked to the beads. The beads were then washed two times with 25 μL of 10 mM TrisHCl (pH 8) and once with 10 μL of water. The biotinylated strands were then eluted from the beads with 12 μL of 2 M NH[0391] 4OH at 60° C. for 10 minutes. The backbone of the DNA was cleaved by incubating the samples for 10 min at 95° C. (with a closed lid), and ammonia was evaporated from the samples by incubating the samples for 11 min at 80° C.
  • The cleavage fragments were then analyzed by MALDI-TOF mass spectrometry as described in Example 4. The T-allele generated a unique fragment of 3254 Daltons. The C-allele generated a unique fragment of 4788 Daltons. These fragements were distinguishable in mass spectra. Thus, the above-identified procedure was successfully utilized to genotype individuals heterozygous for the C-allele and T-aliele in UCP-2. [0392]
  • B. Glycosylase Analysis Utilizing Pooled DNA Samples [0393]
  • The glycosylase assay was conducted using pooled samples to detect genetic variability at the UCP-2 locus. DNA of known genotype was pooled from eleven individuals and was diluted to a fixed concentration of 5 ng/μL. The procedure provided in Example 3A was followed using 2 pmol of forward primer having a sequence of 5′-CCCAGTCACGACGTTGTAAAACGTCTTGGCCTTGCAGATCCAAG-3′ (SEQ ID NO: 93) and 15 pmol of reverse primer having the [0394] sequence 5′-AGCGGATAACAATTTCACACAGGCCATCACACCGCGGTACTG-3′ (SEQ ID NO: 94). In addition, 5 pmol of biotinylated primer having the sequence 5′bioCCCAGTCACGACGTTGTAAAACG 3′ (SEQ ID NO: 97) can be introduced to the PCR reaction after about two cycles. The fragments were analyzed via MALDI-TOF mass spectroscopy (Example 4). As determined in Example 3A, the T-allele, which generated a unique fragment of 3254 Daltons, could be distinguished in mass spectra from the C-allele, which generated a unique fragment of 4788 Daltons. Allelic frequency in the pooled samples was quantified by integrating the area under each signal corresponding to an allelic fragment. Integration was accomplished by hand calculations using equations well known to those skilled in the art. In the pool of eleven samples, this procedure suggested that 40.9% of the individuals harbored the T allele and 59.09% of the individuals harbored the C allele.
  • C. Glycosylase-Mediated Microsatellite Analysis [0395]
  • A glycosylase procedure was utilized to identify microsatellites of the Bradykinin Receptor 2 (BKR-2) sequence. The sequence for BKR-2 is deposited in GenBank under accession number X86173. BKR-2 includes a SNP in the promoter region, which is a C to T variation, as well as a SNP in a repeated unit, which is a G to T variation. The procedure provided in Example 3A was utilized to identify the SNP in the promotor region, the SNP in the microsattelite repeat region, and the number of repeated units in the microsattelite region of BKR-2. Specifically, a forward PCR primer having the [0396] sequence 5′-CTCCAGCTGGGCAGGAGTGC-3′ (SEQ ID NO: 95) and a reverse primer having the sequence 5′-CACTTCAGTCGCTCCCT-3′ (SEQ ID NO: 96) were utilized to amplify BKR-2 DNA in the presence of uracil. The amplicon was fragmented by UDG followed by backbone cleavage. The cleavage fragments were analyzed by MALDI-TOF mass spectrometry as described in Example 4.
  • With regard to the SNP in the BKR-2 promotor region having a C to T variation, the C-allele generated a unique fragment having a mass of 7342.4 Daltons, and the T-allele generated a unique fragment having a mass of 7053.2 Daltons. These fragments were distinguishable in mass spectra. Thus, the above-identified procedure was successfully utilized to genotype individuals heterozygous for the C-allele and T-allele in the promotor region of BKR-2. [0397]
  • With regard to the SNP in the BKR-2 repeat region having a G to T variation, the T-allele generated a unique fragment having a mass of 1784 Daltons, which was readily detected in a mass spectrum. Hence, the presence of the T-allele was indicative of the G to T sequence variation in the repeat region of BKR-2. [0398]
  • In addition, the number of repeat regions was distinguished between individuals having two repeat sequences and individuals having three repeat sequences in BKR-2. The DNA of these individuals did not harbor the G to T sequence variation in the repeat sequence as each repeat sequence contained a G at the SNP locus. The number of repeat regions was determined in individual samples by calculating the area under a signal corresponding to a unique DNA fragment having a mass of 2771.6 Daltons. This signal in spectra generated from individuals having two repeat regions had an area that was thirty-three percent less than the area under the same signal in spectra generated from individuals having three repeat regions. Thus, the procedures discussed above can be utilized to genotype individuals for the number of repeat sequences present in BKR-2. [0399]
  • D. Bisulfite Treatment Coupled with Glycosylase Digestion [0400]
  • Bisulfite treatment of genomic DNA can be utilized to analyze positions of methylated cytosine residues within the DNA. Treating nucleic acids with bisulfite deaminates cytosine residues to uracil residues, while methylated cytosine remains unmodified. Thus, by comparing the sequence of a PCR product generated from genomic DNA that is not treated with bisulfite with the sequence of a PCR product generated from genomic DNA that is treated with bisulfite, the degree of methylation in a nucleic acid as well as the positions where cytosine is methylated can be deduced. [0401]
  • Genomic DNA (2 μg) was digested by incubation with 1 μL of a restriction enzyme at 37° C. for 2 hours. An aliquot of 3 M NaOH was added to yield a final concentration of 0.3M NaOH in the digestion solution. The reaction was incubated at 37° C. for 15 minutes followed by treatment with 5.35M urea, 4.44M bisulfite, and 10 mM hydroquinone, where the final concentration of hydroquinone is 0.5 mM. [0402]
  • The sample that was treated with bisulfite (sample A) was compared to the same digestion sample that had not undergone bisulfite treatment (sample B). After sample A was treated with bisulfite as described above, sample A and sample B were amplified by a standard PCR procedure. The PCR procedure included the step of overlaying each sample with mineral oil and then subjecting the sample to thermocycling (20 cycles of 15 minutes at 55° C. followed by 30 seconds at 95° C.). The PCR reaction contained four nucleotide bases, C, A, G, and U. The mineral oil was removed from each sample, and the PCR products were purified with glassmilk. Sodium iodide (3 volumes) and glassmilk (5 μL) were added to samples A and B. The samples were then placed on ice for 8 minutes, washed with 420 μL cold buffer, centrifuged for 10 seconds, and the supernatant fractions were removed. This process was repeated twice and then 25 μL of water was added. Samples were incubated for 5 minutes at 37° C., were centrifuged for 20 seconds, and the supernatant fraction was collected, and then this incubation/centrifugation/supernatant fraction collection procedure was repeated. 50 μL 0.1 M NaOH was then added to the samples to denature the DNA. The samples were incubated at room temperature for 5 minutes, washed three times with 50 μL of 10 mM TrisHCl (pH 8), and resuspended in 10 [0403] μL 60 mM TrisHCl/1 mM EDTA, pH 7.9.
  • The sequence of PCR products from sample A and sample B were then treated with 2U of UDG (MBI Fermentas) and then subjected to backbone cleavage, as described herein. The resulting fragments from each of sample A and sample B were analyzed by MALDI-TOF mass spectroscopy as described in Example 4. Sample A gave rise to a greater number of fragments than the number of fragments arising from sample B, indicative that the nucleic acid harbored at least one methylated cytosine moiety. [0404]
  • EXAMPLE 7
  • Fen-Ligase-Mediated Haplotyping [0405]
  • Haplotyping procedures permit the selection of a fragment from one of an individual's two homologous chromosomes and to genotype linked SNPs on that fragment. The direct resolution of haplotypes can yield increased information content, improving the diagnosis of any linked disease genes or identifying linkages associated with those diseases. In previous studies, haplotypes were typically reconstructed indirectly through pedigree analysis (in cases where pedigrees were available) through laborious and unreliable allele-specific PCR or through single-molecule dilution methods well known in the art. [0406]
  • A haplotyping procedure was used to determine the presence of two SNPs, referred to as SNP1 and SNP2, located on one strand in a DNA sample. The haplotyping procedure used in this assay utilized Fen-1, a site-specific “flap” endonuclease that cleaves DNA “flaps” created by the overlap of two oligonucleotides hybridized to a target DNA strand. The two overlapping oligonucleotides in this example were short arm and long arm allele-specific adaptors. The target DNA was an amplified nucleic acid that had been denatured and contained SNP1 and SNP2. [0407]
  • The short arm adaptor included a unique sequence not found in the target DNA. The 3′ distal nucleotide of the short arm adaptor was identical to one of the SNP1 alleles. Moreover, the long arm adaptor included two regions: a 3′ region complementary to the short arm and a 5′ gene-specific region complementary to the fragment of interest adjacent to the SNP. If there was a match between the adaptor and one of the homologues, the Fen enzyme recognized and cleaved the overlapping flap. The short arm of the adaptor was then ligated to the remainder of the target fragment (minus the SNP site). This ligated fragment was used as the forward primer for a second PCR reaction in which only the ligated homologue was amplified. The second PCR product (PCR2) was then analyzed by mass spectrometry. If there was no match between the adaptors and the target DNA, there was no overlap, no cleavage by Fen-1, and thus no PCR2 product of interest. [0408]
  • If there was more than one SNP in the sequence of interest, the second SNP (SNP2) was found by using an adaptor that was specific for SNP2 and hybridizing the adaptor to the PCR2 product containing the first SNP. The Fen-ligase and amplification procedures were repeated for the PCR2 product containing the first SNP. If the amplified product yielded a second SNP, then SNP1 and SNP2 were on the same fragment. [0409]
  • If the SNP is unknown, then four allele-specific adaptors (e.g. C, G, A, and T) can be used to hybridize with the target DNA. The substrates are then treated with the Fen-ligase protocol, including amplification. The PCR2 products can be analyzed by PROBE, as described herein, to determine which adaptors were hybridized to the DNA target and thus identify the SNPs in the sequence. [0410]
  • A Fen-ligase assay was used to detect two SNPs present in Factor VII. These SNPs are located 814 base pairs apart from each other. SNP1 was located at position 8401 (C to T), and SNP2 was located at 9215 (G to A). [0411]
  • A. First Amplification Step [0412]
  • A PCR product (PCR1) was generated for a known heterozygous individual at SNP1, a short distance from the 5′ end of the SNP. Specifically, a 10 μL PCR reaction was performed by mixing 1.5 mM MgCl[0413] 2, 200 μM of each dNTP, 0.5 U HotStar polymerase, 0.1 μM of a forward primer having the sequence 5′-GCG CTC CTG TCG GTG CCA (SEQ ID NO: 56), 0.1 μM of a reverse primer having the sequence 5′-GCC TGA CTG GTG GGG CCC (SEQ ID NO: 57), and 1 ng of genomic DNA. The annealing temperature was 58° C., and the amplification process yielded fragments that were 861 bp in length.
  • The PCR1 reaction mixture was divided in half and was treated with an [0414] exonuclease 1/SAP mixture (0.22 μL mixture/5 μL PCR1 reaction) which contained 1.0 μL SAP and 0.1 μL exon1. The exonuclease treatment was done for 30 minutes at 37° C. and then 20 minutes at 85° C. to denature the DNA.
  • B. Adaptor Oligonucleotides [0415]
  • A solution of allele-specific adaptors (C and T), containing of one long and one short oligonucleotide per adaptor, was prepared. The long arm and short arm oligonucleotides of each adaptor (10 μM) were mixed in a 1:1 ratio and heated for 30 seconds at 95° C. The temperature was reduced in 2° C. increments to 37° C. for annealing. The C-adaptor had a short arm sequence of 5′-CAT GCA TGC ACG GTC (SEQ ID NO: 58) and a long arm sequence of 5′-CAG AGA GTA CCC CTC GAC CGT GCA TGC ATG (SEQ ID NO: 59). Hence, the long arm of the adaptor was 30 bp (15 bp gene-specific), and the short arm was 15 bp. The T-adaptor had a short arm sequence of 5′-CAT GCA TGC ACG GTT (SEQ ID NO: 60) and a long arm sequence of 5′-GTA CGT ACG TGC CAA CTC CCC ATG AGA GAC (SEQ ID NO: 61). The adaptor could also have a hairpin structure in which the short and long arm are separated by a loop containing of 3 to 10 nucleotides (SEQ ID NO: 118). [0416]
  • C. FEN-ligase Reaction [0417]
  • In two tubes (one tube for each allele-specific adaptor per sample) was placed a solution (Solution A) containing of 3.5 μl 10 mM 16% PEG/50 mM MOPS, 1.2 μl 25 mM MgCl[0418] 2, 1.5 μl 10X Ampligase Buffer, and 2.5 μl PCR1. Each tube containing Solution A was incubated at 95° C. for 5 minutes to denature the PCR1 product. A second solution (Solution B) containing of 1.65 μl Ampligase (Thermostable ligase, Epicentre Technologies), 1.65 μl 200 ng/μl MFEN (from Methanocuccus jannaschil), and 3.0 μl of an allel specific adaptor (C or T) was prepared. Thus, different variations of Solution B, each variation containing of different allele-specific adaptors, were made. Solution B was added to Solution A at 95° C. and incubated at 55° C. for 3 hours. The total reaction volume was 15.0 μl per adaptor-specific reaction. For a bi-allelic system, 2×15.0 μl reactions were required.
  • The Fen-ligase reaction in each tube was then deactivated by adding 8.0 μl 10 mM EDTA. Then, 1.0 μl exoIII/Buffer (70%/30%) solution was added to each sample and incubated 30 minutes at 37° C., 20 minutes at 70° C. (to deactivate exoIII), and 5 minutes at 95° C. (to denature the sample and dissociate unused adaptor from template). The samples were cooled in an ice slurry and purified on UltraClean PCR Clean-up (MoBio) spin columns which removed all fragments less than 100 base pairs in length. The fragments were eluted with 50 μl H[0419] 2O.
  • D. Second Amplification Step [0420]
  • A second amplification reaction (PCR2) was conducted in each sample tube using the short arm adaptor (C or T) sequence as the forward primer (minus the SNP1 site). Only the ligated homologue was amplified. A standard PCR reaction was conducted with a total volume of 10.0 μl containing of 1×Buffer (final concentration), 1.5 mM final concentration MgCl[0421] 2, 200 μM final concentration dNTPs, 0.5 U HotStar polymerase, 0.1 μM final concentration forward primer 5′-CAT GCA TGC ACG GT (SEQ ID NO: 62), 0.1 μM final concentration reverse primer 5′-GCC TGA CTG GTG GGG CCC (SEQ ID NO: 63), and 1.0 μl of the purified FEN-ligase reaction solution. The annealing temperature was 58° C. The PCR2 product was analyzed by MALDI TOF mass spectroscopy as described in Example 4. The mass spectrum of Fen SNP1 showed a mass of 6084.08 Daltons, representing the C allele.
  • E. Genotyping Additional SNPs [0422]
  • The second SNP (SNP2) can be found by using an adaptor that is specific for SNP2 and hybridizing that adaptor to the PCR2 product containing the first SNP. The Fen-ligase and amplification procedures are repeated for the PCR2 product containing the first SNP. If the amplified product yields a second SNP, then SN1 and SN2 are on the same fragment. The mass spectrum of SNP2, representing the T allele, showed a mass of 6359.88 Daltons. [0423]
  • This assay also can be performed upon pooled DNA to yield haplotype frequencies as described herein. The Fen-ligase assay can be used to analyze multiplexes as described herein. [0424]
  • EXAMPLE 8
  • Nickase-Mediated Sequence Analysis [0425]
  • A DNA nickase, or DNase, was used to recognize and cleave one strand of a DNA duplex. NY2A nickase and NYS1 nickase (Megabase), which cleave DNA at the following sites: [0426]
  • NY2A: 5′ . . . R AG . . . 3′[0427]
  • 3[0428] 40 . . . Y⇓TC . . . 5′ where R=A or G and Y=C or T
  • NYS1: 5′ . . . ⇓CC[A/G/T] . . . 3′[0429]
  • 3′ . . . GG[T/C/A] . . . 5′[0430]
  • were used. [0431]
  • A. Nickase Digestion [0432]
  • Tris-HCl (10 mM), KCl (10 mM, pH 8.3), magnesium acetate (25 mM), BSA (1 mg/mL), and 6 U of Cvi NY2A or Cvi NYS1 Nickase (Megabase Research) were added to 25 pmol of double-stranded oligonucleotide template having a sequence of 5′-CGC AGG GTT TCC TCG TCG CAC TGG GCA TGT G-3′ (SEQ ID NO: 90, Operon, Alameda, Calif.) synthesized using standard phosphoramidite chemistry. With a total volume of 20 μL, the reaction mixture was incubated at 37° C. for 5 hours, and the digestion products were purified using ZipTips (Millipore, Bedford, Mass.) as described in Example 5. The samples were analyzed by MALDI-TOF mass spectroscopy as described in Example 1. The nickase Cvi NY2A yielded three fragments with masses 4049.76 Daltons, 5473.14 Daltons, and 9540.71 Daltons. The Cvi NYS1 nickase yielded fragments with masses 2063.18 Daltons, 3056.48 Daltons, 6492.81 Daltons, and 7450.14 Daltons. [0433]
  • B. Nickase Digestion of Pooled Samples [0434]
  • DQA (HLA ClassII-DQ Alpha, expected fragment size=225 bp) was amplified from the genomic DNA of 100 healthy individuals. DQA was amplified using standard PCR chemistry in a reaction having a total volume of 50 μL containing of 10 mM Tris-HCl, 10 mM KCl (pH 8.3), 2.5 mM MgCl[0435] 2, 200 μM of each dNTP, 10 pmol of a forward primer having the sequence 5′-GTG CTG CAG GTG TAA ACT TGT ACC AG-3′(SEQ ID NO: 64), 10 pmol of a reverse primer having the sequence 5′-CAC GGA TCC GGT AGC AGC GGT AGA GTT G-3′(SEQ ID NO: 65), 1 U DNA polymerase (Stoffel fragment, Perkin Elmer), and 200 ng human genomic DNA (2 ng DNA/individual). The template was denatured at 94° C. for 5 minutes. Thermal cycling was continued with a touch-down program that included 45 cycles of 20 seconds at 94° C., 30 seconds at 56° C., 1 minute at 72° C., and a final extension of 3 minutes at 72° C. The crude PCR product was used in the subsequent nickase reaction.
  • The unpurified PCR product was subjected to nickase digestion. Tris-HCl (10 mM), KCl (10 mM, pH 8.3), magnesium acetate (25 mM), BSA (1 mg/mL), and 5 U of Cvi NY2A or Cvi NYS1 Nickase (Megabase Research) were added to 25 pmol of the amplified template with a total reaction volume of 20 μL. The mixture was then incubated at 37° C. for 5 hours. The digestion products were purified with either ZipTips (Millipore, Bedford, Mass.) as described in Example 5. The samples were analyzed by MALDI-TOF mass spectroscopy as described in Example 4. This assay also can be used to do multiplexing and standardless genotyping as described herein. [0436]
  • To simplify the nickase mass spectrum, the two complementary strands can be separated after digestion by using a single-stranded undigested PCR product as a capture probe. This probe (preparation shown below in Example 8C) can be hybridized to the nickase fragments in hybridization buffer containing 200 mM sodium citrate and 1% blocking reagent (Boehringer Mannheim). The reaction is heated to 95° C. for 5 minutes and cooled to room temperature over 30 minutes by using a thermal cycler (PTC-200 DNA engine, MJ Research, Waltham, Mass.). The capture probe-nickase fragment is immobilized on 140 μg of streptavidin-coated magnetic beads. The beads are subsequently washed three times with 70 mM ammonium citrate. The captured single-stranded nickase fragments are eluted by heating to 80° C. for 5 minutes in 5 μL of 50 mM ammonium hydroxide. [0437]
  • C. Preparation of Capture Probe [0438]
  • The capture probe is prepared by amplifying the human β-globin gene (3′ end of [0439] intron 1 to 5′ end of exon 2) via PCR methods in a total volume of 50 μL containing of GeneAmp 1XPCR Buffer II, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTP mix, 10 pmol of each primer (forward primer 5′-ACTGGGCATGTGGAGACAG-3′(SEQ ID NO: 66) and biotinylated reverse primer bio5′-GCACTTTCTTGCCATGAG-3′(SEQ ID: 67), 2 U of AmpliTaq Gold, and 200 ng of human genomic DNA. The template is denatured at 94° C. for 8 minutes. Thermal cycling is continued with a touch-down program that included 11 cycles of 20 seconds at 94° C., 30 seconds at 64° C., 1 minute at 72° C.; and a final extension of 5 minutes at 72° C. The amplicon is purified using UltraClean™ PCR clean-up kit (MO Bio Laboratories, Solano Beach, Calif.).
  • EXAMPLE 9
  • Multiplex Type IIS SNP Assay [0440]
  • A Type IIS assay was used to identify human gene sequences with known SNPs. The Type IIS enzyme used in this assay was Fok I which effected double-stranded cleavage of the target DNA. The assay involved the steps of amplification and Fok I treatment of the amplicon. In the amplification step, the primers were designed so that each PCR product of a designated gene target was less than 100 bases such that a Fok I recognition sequence was incorporated at the 5′ and 3′ end of the amplicon. Therefore, the fragments that were cleaved by Fok I included a center fragment containing the SNP of interest. [0441]
  • Ten human gene targets with known SNPs were analyzed by this assay. Sequences of the ten gene targets, as well as the primers used to amplify the target regions, are found in Table 5. The ten targets were lipoprotein lipase, prothrombin, factor V, cholesterol ester transfer protein (CETP), factor VII, factor XIII, HLA-[0442] H exon 2, HLA-H exon 4, methylenetetrahydrofolate reductase (MTHR), and P53 exon 4 codon 72.
  • Amplification of the ten human gene sequences were carried out in a single 50 μL volume PCR reaction with 20 ng of human genomic DNA template in 5 PCR reaction tubes. Each reaction vial contained 1×PCR buffer (Qiagen), 200 μM dNTPs, 1 U Hotstar Taq polymerase (Qiagen), 4 mM MgCl[0443] 2, and 10 pmol of each primer. US8, having sequence of 5′TCAGTCACGACGTT3′(SEQ ID NO: 68), and US9, having sequence of 5′CGGATAACAATTTC3′(SEQ ID NO: 69), were used for the forward and reverse primers respectively. Moreover, the primers were designed such that a Fok I recognition site was incorporated at the 5′ and 3′ ends of the amplicon. Thermal cycling was performed in 0.2 mL tubes or a 96 well plate using a MJ Research Thermal Cycler (calculated temperature) with the following cycling parameters: 94° C. for 5 minutes; 45 cycles: 94° C. for 20 seconds, 56° C. for 20 seconds, 72° C. for 60 seconds; and 72° C. for 3 minutes.
  • Following PCR, the sample was treated with 0.2 U Exonuclease I (Amersham Pharmacia) and S Alkaline Phosphotase (Amersham Pharmacia) to remove the unincorporated primers and dNTPs. Typically, 0.2 U of exonuclease I and SAP were added to 5 μL of the PCR sample. The sample was then incubated at 37° C. for 15 minutes. Exonuclease I and SAP were then inactivated by heating the sample up to 85° C. for 15 minutes. Fok I digestion was performed by adding 2 U of Fok I (New England Biolab) to the 5 uL PCR sample and incubating at 37° C. for 30 minutes. Since the Fok I restriction sites are located on both sides of the amplicon, the 5′ and 3′ cutoff fragments have higher masses than the center fragment containing the SNP. The sample was then purified by anion exchange and analyzed by MALDI-TOF mass spectrometry as described in Example 4. The masses of the gene fragments from this multiplexing experiment are listed in Table 6. These gene fragments were resolved in mass spectra thereby allowing multiplex analysis of sequence variability in these genes. [0444]
    TABLE 5
    Genes for Multiplex Type IIS Assay
    Seq. ID Seq.
    Gene Sequence No Primers ID No.
    Lipoprotein cctttgagaa agggctctgc ttgagttgta   98-99 5′ 70
    Lipase gaaagaaccg ctgcaacaat caatttcatcgctggatgcaatct
    (Asn291Ser) ctgggctatg agatca[a
    Figure US20030190644A1-20031009-P00801
    g]taa agtcagagcc
    gggctatgagatc 3′
    aaaagaagca gcaaaatgta
    5′ 71
    caatttcacacagcggatgcttct
    tttggctctgact 3′
    Prothrombin 26731 gaattatttt tgtgtttcta aaactatggt 100- 5′ 72
    tcccaataaa agtgactctc 101 tcagtcacgacgttggatgccaa
    26781 agc[g
    Figure US20030190644A1-20031009-P00801
    a]agcctc aatgctccca
    taaaagtgactctcagc 3′
    gtgctattca tgggcagctc tctgggctca
    5′ 73
    cggataacaatttcggatgcact
    gggagcattgaggc 3′
    Factor V taataggact acttctaatc tgtaagagca 102- 5′ 74
    (Arg506Gln) gatccctgga caggc[g
    Figure US20030190644A1-20031009-P00801
    a]agga
    103 tcagtcacgacgttggatgagca
    gatccctggacaggc 3′
    atacaggtat tttgtccttg aagtaacctt tcag 5′ 75
    cggataacaatttcggatggaca
    aaatacctgtattcc 3′
    Cholesterol ester 1261 ctcaccatgg gcatttgatt gcagagcage 104- 5′ 76
    transfer protein tccgagtcc[g
    Figure US20030190644A1-20031009-P00801
    a] tccagagctt
    105 tcagtcacgacgttggatgcaga
    (CETP) (I405V) gcagctccgagtc 3′
    1311 cctgcagtca atgatcaccg ctgtgggcat 5′ 77
    ccctgaggtc atgtctcgta cagcggtgatcattggatgcagg
    aagctctgg 3′
    Factor VII 1221 agcaaggact cctgcaaggg ggacagtgga 106- 5′ 78
    (R353Q) ggcccacatg ccacccacta 107 tcagtcacgacgttggatgccca
    catgccacccactac 3′
    1271 cc[a
    Figure US20030190644A1-20031009-P00801
    g]gggcacg tggtacctga
    5′ 79
    cgggcatcgt cagctggggc cagggctgcg cggataacaatttcggatgcccg
    tcaggtaccacg 3′
    Factor XIII 111 caataactct aatgcagcgg aagatgacct 108- 5′ 80
    (V34L) gcccacagtg gagcttcagg 109 tcagtcacgacgttggatgccca
    cagtggagcttcag 3′
    161 gc[g
    Figure US20030190644A1-20031009-P00801
    t]tggtgcc ccggggcgtc
    5′ 81
    aacctgcaag gtatgagcat accccccttc gctcataccttgcaggatgacg
    3′
    HLA-H exon 2 361 ttgaagctttgggctacgtg gatgaccagc 110- 5′ 82
    (His63Asp) tgttcgtgtt ctatgat[c
    Figure US20030190644A1-20031009-P00801
    g]at
    111 tcagtcacgacgttggatgacca
    gctgttcgtgttc 3′
    411 gagagtcgcc gtgtggagcc ccgaactcca 5′ 83
    tgggtttcca gtagaatttc tacatggagttcggggatgcaca
    cggcgactctc 3′
    HLA-H exon 4 1021 ggataacctt ggctgtaccc cctggggaag 112- 5′ 84
    (Cys282Tyr) agcagagata tacgt[g
    Figure US20030190644A1-20031009-P00801
    a]ccag
    113 tcagtcacgacgttggatgggga
    agagcagagatatacgt 3′
    1071 gtggagcacc caggcctgga tcagcccctc 5′ 85
    attgtgatct gggagccctc gaggggctgatccaggatgggt
    gctccac 3′
    Methylentetra- 761 tgaagcactt gaagga gaag gtgtctgcgg 114- 5′ 86
    hydrofolate- gag[c
    Figure US20030190644A1-20031009-P00801
    t]cgattt catcatcacg
    115 tcagtcacgacgttggatgggga
    redctase agagcagagatatacgt 3′
    (MTHR)
    (Ala222Val)
    811 cagcttttct ttgaggctga cacattcttc 5′ 87
    gaggggctgatccaggatgggt
    gctccac 3′
    P53 Exon4 12101 tccagatgaa gctcccagaa 116- 5′ 88
    Codon 72 tgccagaggc tgctcccc[g
    Figure US20030190644A1-20031009-P00801
    c]c gtggcccctg
    117 gatgaagctcccaggatgccag
    (Arg72Pro) aggc 3′
    12151 caccagcagc tcctacaccg 5′ 89
    gcggcccctg gccgccggtgtaggatgctgctg
    gtgc 3′
  • [0445]
    TABLE 6
    The mass of Center Fragments for Ten Different SNP Typing by IIS Assay
    Gene LPL(Asn291Ser) Prothrombin FV(Arg506Gln) CETP(I405V) FVII(R353Q) FXIII(V34)
    Genotype A G G A G A G A G A G T
    + strand 6213 6229 5845 5829 5677 5661 3388 3372 6128 6112 5058 5033
    mass
    (Da)
    − strand 6129 6114 5949 5964 5472 5487 3437 3452 6174 6189 4916 4940
    mass
    (Da)
    Gene Hlah2 Hlah4 MTHR(Ala222Val) P53exon4(Arg72Pro)
    Genotype C G G A C T G C
    + strand 5889 5929 4392 4376 4400 4415 4586 4546
    mass
    (Da)
    − strand 5836 5796 4319 4334 4368 4352 4724 4764
    mass
    (Da)
  • EXAMPLE 10
  • Exemplary use of Parental Medical History Parameter for Stratification of Healthy Datebase [0446]
  • A healthy database can be used to associate a disease state with a specific allele (SNP) that has been found to show a strong association between age and the allele, in particular the homozygous genotype. The method involves using the same healthy database used to identify the age dependent association, however stratification is by information given by the donors about common disorders from which their parents suffered (the donor's familial history of disease). There are three possible answers a donor could give about the health status of their parents: neither were affected, one was affected or both were affected. Only donors above a certain minimum age, depending on the disease, are utilized, as the donors parents must be old enough to to have exhibited clinical disease phenotypes. The genotype frequency in each of these groups is determined and compared with each other. If there is an association of the marker in the donor to a disease the frequency of the heterozyous genotype will be increased. The frequency of the homozygous genotype should not increase, as it should be significantly underrepresented in the healthy population. [0447]
  • EXAMPLE 11
  • Method and Device for Identifying a Biological Sample Description [0448]
  • A method and device for identifying a biological sample is provided. Referring now to FIG. 24, an [0449] apparatus 10 for identifying a biological sample is disclosed. The apparatus 10 for identifying a biological sample generally comprises a mass spectrometer 15 communicating with a computing device 20. In an embodiment, the mass spectrometer can be a MALDI-TOF mass spectrometer manufactured by Bruker-Franzen Analytik GmbH; however, it will be appreciated that other mass spectrometers can be substituted. The computing device 20 is typically a general purpose computing device. It will be appreciated that the computing device could be alternatively configured, for example, it can be integrated with the mass spectrometer or could be part of a computer in a larger network system.
  • The [0450] apparatus 10 for identifying a biological sample can operate as an automated identification system having a robot 25 with a robotic arm 27 configured to deliver a sample plate 29 into a receiving area 31 of the mass spectrometer 15. In such a manner, the sample to be identified can be placed on the plate 29 and automatically received into the mass spectrometer 15. The biological sample is then processed in the mass spectrometer to generate data indicative of the mass of DNA fragments in the biological sample. This data can be sent directly to computing device 20, or can have some preprocessing or filtering performed within the mass spectrometer. In an embodiment, the mass spectrometer 15 transmits unprocessed and unfiltered mass spectrometry data to the computing device 20. It will be appreciated that the analysis in the computing device can be adjusted to accommodate preprocessing or filtering performed within the mass spectrometer.
  • Referring now to FIG. 25, a [0451] general method 35 for identifying a biological sample is shown. In method 35, data are received into a computing device from a test instrument in block 40. Generally the data are received in a raw, unprocessed and unfiltered form, but alternatively can have some form of filtering or processing applied. The test instrument of an exemplary embodiment is a mass spectrometer as described above. It will be appreciated that other test instruments could be substituted for the mass spectrometer.
  • The data generated by the test instrument, and in particular the mass spectrometer, includes information indicative of the identification of the biological sample. More specifically, the data are indicative of the DNA composition of the biological sample. Typically, mass spectrometry data gathered from DNA samples obtained from DNA amplification techniques are noisier than, for example, those from typical protein samples. This is due in part because protein samples are more readily prepared in more abundance, and protein samples are more easily ionizable as compared to DNA samples. Accordingly, conventional mass spectrometer data analysis techniques are generally ineffective for DNA analysis of a biological sample. To improve the analysis capability so that DNA composition data can be more readily discerned, an embodiment uses wavelet technology for analyzing the DNA mass spectrometry data. Wavelets are an analytical tool for signal processing, numerical analysis, and mathematical modeling. Wavelet technology provides a basic expansion function which is applied to a data set. Using wavelet decomposition, the data set can be simultaneously analyzed in the time and frequency domains. Wavelet transformation is the technique of choice in the analysis of data that exhibit complicated time (mass) and frequency domain information, such as MALDI-TOF DNA data. Wavelet transforms as described herein have superior denoising properties as compared to conventional Fourier analysis techniques. Wavelet transformation has proven to be particularly effective in interpreting the inherently noisy MALDI-TOF spectra of DNA samples. In using wavelets, a “small wave” or “scaling function” is used to transform a data set into stages, with each stage representing a frequency component in the data set. Using wavelet transformation, mass spectrometry data can be processed, filtered, and analyzed with sufficient discrimination to be useful for identification of the DNA composition for a biological sample. [0452]
  • Referring again to FIG. 25, the data received in [0453] block 40 is denoised in block 45. The denoised data then has a baseline correction applied in block 50. A baseline correction is generally necessary as data coming from the test instrument, in particular a mass spectrometer instrument, has data arranged in a generally exponentially decaying manner. This generally exponential decaying arrangement is not due to the composition of the biological sample, but is a result of the physical properties and characteristics of the test instrument, and other chemicals involved in DNA sample preparation. Accordingly, baseline correction substantially corrects the data to remove a component of the data attributable to the test system, and sample preparation characteristics.
  • After denoising in [0454] block 45 and the baseline correction in block 50, a signal remains which is generally indicative of the composition of the biological sample. Due to the extraordinary discrimination required for analyzing the DNA composition of the biological sample, the composition is not readily apparent from the denoised and corrected signal. For example, although the signal can include peak areas, it is not yet clear whether these “putative” peaks actually represent a DNA composition, or whether the putative peaks are the result of a systemic or chemical aberration. Further, any call of the composition of the biological sample would have a probability of error which would be unacceptable for clinical or therapeutic purposes. In such critical situations, there needs to be a high degree of certainty that any call or identification of the sample is accurate. Therefore, additional data processing and interpretation is necessary before the sample can be accurately and confidently identified.
  • Since the quantity of data resulting from each mass spectrometry test is typically thousands of data points, and an automated system can be set to perform hundreds or even thousands of tests per hour, the quantity of mass spectrometry data generated is enormous. To facilitate efficient transmission and storage of the mass spectrometry data, block 55 shows that the denoised and baseline corrected data are compressed. [0455]
  • In one embodiment, the biological sample is selected and processed to have only a limited range of possible compositions. Accordingly, it is therefore known where peaks indicating composition should be located, if present. Taking advantage of knowing the location of these expected peaks, in [0456] block 60 the method 35 matches putative peaks in the processed signal to the location of the expected peaks. In such a manner, the probability of each putative peak in the data being an actual peak indicative of the composition of the biological sample can be determined. Once the probability of each peak is determined in block 60, then in block 65 the method 35 statistically determines the composition of the biological sample, and determines if confidence is high enough to calling a genotype.
  • Referring again to block 40, data are received from the test instrument, which can be a mass spectrometer. In a specific illustration, FIG. 26 shows an example of data from a mass spectrometer. The [0457] mass spectrometer data 70 generally comprises data points distributed along an x-axis 71 and a y-axis 72. The x-axis 71 represents the mass of particles detected, while the y-axis 72 represents a numerical concentration of the particles. As can be seen in FIG. 26, the mass spectrometry data 70 is generally exponentially decaying with data at the left end of the x-axis 73 generally decaying in an exponential manner toward data at the heavier end 74 of the x-axis 71. The general exponential presentation of the data is not indicative of the composition of the biological sample, but is more reflective of systematic error and characteristics. Further, as described above and illustrated in FIG. 26, considerable noise exists in the mass spectrometry DNA data 70.
  • Referring again to block 45, where the raw data received in [0458] block 40 is denoised, the denoising process will be described in more detail. As illustrated in FIG. 25, the denoising process generally entails 1) performing a wavelet transformation on the raw data to decompose the raw data into wavelet stage coefficients; 2) generating a noise profile from the highest stage of wavelet coefficients; and 3) applying a scaled noise profile to other stages in the wavelet transformation. Each step of the denoising process is further described below.
  • Referring now to FIG. 27, the wavelet transformation of the raw mass spectrometry data is generally diagramed. Using wavelet transformation techniques, the [0459] mass spectrometry data 70 is sequentially transformed into stages. In each stage, the data are represented in a high stage and a low stage, with the low stage acting as the input to the next sequential stage. For example, the mass spectrometry data 70 is transformed into stage 0 high data 82 and stage 0 low data 83. The stage 0 low data 83 is then used as an input to the next level transformation to generate stage 1 high data 84 and stage 1 low data 85. In a similar manner, the stage 1 low data 85 is used as an input to be transformed into stage 2 high data 86 and stage 2 low data 87. The transformation is continued until no more useful information can be derived by further wavelet transformation. For example, in the one embodiment a 24-point wavelet is used. More particularly a wavelet commonly referred to as the Daubechies 24 is used to decompose the raw data. It will be appreciated that other wavelets can be used for the wavelet transformation. Since each stage in a wavelet transformation has one-half the data points of the previous stage, the wavelet transformation can be continued until the stage n low data 89 has around 50 points. Accordingly, the stage n high 88 would contain about 100 data points. Since the exemplary wavelet is 24 points long, little data or information can be derived by continuing the wavelet transformation on a data set of around 50 points.
  • FIG. 28 shows an example of [0460] stage 0 high data 95. Since stage 0 high data 95 is generally indicative of the highest frequencies in the mass spectrometry data, stage 0 high data 95 will closely relate to the quantity of high frequency noise in the mass spectrometry data. In FIG. 29, an exponential fitting formula has been applied to the stage 0 high data 95 to generate a stage 0 noise profile 97. In particular, the exponential fitting formula is in the format A0+A1 EXP (−A2 m). It will be appreciated that other exponential fitting formulae or other types of curve fits can be used.
  • Referring now to FIG. 30, noise profiles for the other high stages are determined. Since the later data points in each stage will likely be representative of the level of noise in each stage, only the later data points in each stage are used to generate a standard deviation figure that is representative of the noise content in that particular stage. More particularly, in generating the noise profile for each remaining stage, only the last five percent of the data points in each stage are analyzed to determined a standard deviation number. It will be appreciated that other numbers of points, or alternative methods could be used to generate such a standard deviation figure. [0461]
  • The standard deviation number for each stage is used with the [0462] stage 0 noise profile (the exponential curve) 97 to generate a scaled noise profile for each stage. For example, FIG. 30 shows that stage 1 high data 98 has stage 1 high data 103 with the last five percent of the data points represented by area 99. The points in area 99 are evaluated to determine a standard deviation number indicative of the noise content in stage 1 high data 103. The standard deviation number is then used with the stage 0 noise profile 97 to generate a stage 1 noise profile.
  • In a similar manner, [0463] stage 2 high 100 has stage 2 high data 104 with the last five percent of points represented by area 101. The data points in area 101 are then used to calculate a standard deviation number which is then used to scale the stage 0 noise profile 97 to generate a noise profile for stage 2 data. This same process is continued for each of the stage high data as shown by the stage n high 105. For stage n high 105, stage n high data 108 has the last five percent of data points indicated in area 106. The data points in area 106 are used to determine a standard deviation number for stage n. The stage n standard deviation number is then used with the stage 0 noise profile 97 to generate a noise profile for stage n. Accordingly, each of the high data stages has a noise profile.
  • FIG. 31 shows how the noise profile is applied to the data in each stage. Generally, the noise profile is used to generate a threshold which is applied to the data in each stage. Since the noise profile is already scaled to adjust for the noise content of each stage, calculating a threshold permits further adjustment to tune the quantity of noise removed. Wavelet coefficients below the threshold are ignored while those above the threshold are retained. Accordingly, the remaining data have a substantial portion of the noise content removed. [0464]
  • Due to the characteristics of wavelet transformation, the lower stages, such as [0465] stage 0 and 1, will have more noise content than the later stages such as stage 2 or stage n. Indeed, stage n low data are likely to have little noise at all. Therefore, in an embodiment, the noise profiles are applied more aggressively in the lower stages and less aggressively in the later stages. For example, FIG. 31 shows that stage 0 high threshold is determined by multiplying the stage 0 noise profile by a factor of four. In such a manner, significant numbers of data points in stage 0 high data 95 will be below the threshold and therefore eliminated. Stage 1 high threshold 112 is set at two times the noise profile for the stage 1 high data, and stage 2 high threshold 114 is set equal to the noise profile for stage 2 high. Following this geometric progression, stage n high threshold 116 is therefore determined by scaling the noise profile for each respective stage n high by a factor equal to (½n-2). It will be appreciated that other factors can be applied to scale the noise profile for each stage. For example, the noise profile can be scaled more or less aggressively to accommodate specific systemic characteristics or sample compositions. As indicated above, stage n low data does not have a noise profile applied as stage n low data 118 is assumed to have little or no noise content. After the scaled noise profiles have been applied to each high data stage, the mass spectrometry data 70 has been denoised and is ready for further processing. A wavelet transformation of the denoised signal results in the sparse data set 120 as shown in FIG. 31.
  • Referring again to FIG. 25, the mass spectrometry data received in [0466] block 40 has been denoised in block 45 and is now passed to block 50 for baseline correction. Before performing baseline correction, the artifacts introduced by the wavelet transformation procedure can be removed. Wavelet transformation results vary slightly depending upon which point of the wavelet is used as a starting point. For example, an exemplary embodiment uses the 24-point Daubechies-24 wavelet. By starting the transformation at the 0 point of the wavelet, a slightly different result will be obtained than if starting at points 1 or 2 of the wavelet. Therefore, the denoised data are transformed using every available possible starting point, with the results averaged to determine a final denoised and shifted signal. For example, FIG. 33 shows that the wavelet coefficient is applied 24 different times and then the results averaged to generate the final data set. It will be appreciated that other techniques can be used to accommodate the slight error introduced due to wavelet shifting.
  • The formula 125 is generally indicated in FIG. 33. Once the signal has been denoised and shifted, a denoised and shifted [0467] signal 130 is generated as shown in FIG. 58. FIG. 34 shows an example of the wavelet coefficient 135 data set from the denoised and shifted signal 130.
  • FIG. 36 shows that [0468] putative peak areas 145, 147, and 149 are located in the denoised and shifted signal 150. The putative peak areas are systematically identified by taking a moving average along the signal 150 and identifying sections of the signal 150 which exceed a threshold related to the moving average. It will be appreciated that other methods can be used to identify putative peak areas in the signal 150.
  • [0469] Putative peak areas 145, 147 and 149 are removed from the signal 150 to create a peak-free signal 155 as shown in FIG. 37. The peak-free signal 155 is further analyzed to identify remaining minimum values 157, and the remaining minimum values 157 are connected to generate the peak-free signal 155.
  • FIG. 38 shows a process of using the peak-[0470] free signal 155 to generate a baseline 170 as shown in FIG. 39. As shown in block 162, a wavelet transformation is performed on the peak-free signal 155. All the stages from the wavelet transformation are eliminated in block 164 except for the n low stage. The n low stage will generally indicate the lowest frequency component of the peak-free signal 155 and therefore will generally indicate the system exponential characteristics. Block 166 shows that a signal is reconstructed from the n low coefficients and the baseline signal 170 is generated in block 168.
  • FIG. 39 shows a denoised and shifted data signal 172 positioned adjacent a [0471] correction baseline 170. The baseline correction 170 is subtracted from the denoised and shifted signal 172 to generate a signal 175 having a baseline correction applied as shown in FIG. 40. Although such a denoised, shifted, and corrected signal is sufficient for most identification purposes, the putative peaks in signal 175 are not identifiable with sufficient accuracy or confidence to call the DNA composition of a biological sample.
  • Referring again to FIG. 25, the data from the [0472] baseline correction 50 is now compressed in block 55; the compression technique used in an exemplary embodiment is detailed in FIG. 41. In FIG. 41the data in the baseline corrected data are presented in an array format 182 with x-axis points 183 having an associated data value 184. The x-axis is indexed by the non-zero wavelet coefficients, and the associated value is the value of the wavelet coefficient. In the illustrated data example in table 182, the maximum value 184 is indicated to be 1000. Although a particularly advantageous compression technique for mass spectrometry data is shown, it will be appreciated that other compression techniques can be used. The data also can be stored without compression.
  • In compressing the data according to one embodiment, an [0473] intermediate format 186 is generated. The intermediate format 186 generally comprises a real number having a whole number portion 188 and a decimal portion 190. The whole number portion is the x-axis point 183 while the decimal portion is the value data 184 divided by the maximum data value. For example, in the data 182 a data value “25” is indicated at x-axis point “100” . The intermediate value for this data point would be “100.025”.
  • From the intermediate [0474] compressed data 186 the final compressed data 195 is generated. The first point of the intermediate data file becomes the starting point for the compressed data. Thereafter each data point in the compressed data 195 is calculated as follows: the whole number portion (left of the decimal) is replaced by the difference between the current and the last whole number. The remainder (right of the decimal) remains intact. For example, the starting point of the compressed data 195 is shown to be the same as the intermediate data point which is “100.025”. The comparison between the first intermediate data point “100.025” and the second intermediate data point “150.220” is “50.220”. Therefore, “50.220” becomes the second point of the compressed data 195. In a similar manner, the second intermediate point is “150.220” and the third intermediate data point is “500.0001” . Therefore, the third compressed data becomes “350.000”. The calculation for determining compressed data points is continued until the entire array of data points is converted to a single array of real numbers.
  • FIG. 42 generally describes the method of compressing mass spectrometry data, showing that the data file in [0475] block 201 is presented as an array of coefficients in block 202. The data starting point and maximum is determined as shown in block 203, and the intermediate real numbers are calculated in block 204 as described above. With the intermediate data points generated, the compressed data are generated in block 205. The described compression method is highly advantageous and efficient for compressing data sets such as a processed data set from a mass spectrometry instrument. The method is particularly useful for data, such as mass spectrometry data, that uses large numbers and has been processed to have occasional lengthy gaps in x-axis data. Accordingly, an x-y data array for processed mass spectrometry data can be stored with an effective compression rate of 10×or more. Although the compression technique is applied to mass spectrometry data, it will be appreciated that the method can also advantageously be applied to other data sets.
  • Referring again to FIG. 25, peak heights are now determined in [0476] block 60. The first step in determining peak height is illustrated in FIG. 43 where the signal 210 is shifted left or right to correspond with the position of expected peaks. As the set of possible compositions in the biological sample is known before the mass spectrometry data are generated, the possible positioning of expected peaks is already known. These possible peaks are referred to as expected peaks, such as expected peaks 212, 214, and 216. Due to calibration or other errors in the test instrument data, the entire signal can be shifted left or right from its actual position, therefore, putative peaks located in the signal, such as putative peaks 218, 222, and 224 can be compared to the expected peaks 212, 214, and 216, respectively. The entire signal is then shifted such that the putative peaks align more closely with the expected peaks.
  • Once the putative peaks have been shifted to match expected peaks, the strongest putative peak is identified in FIG. 44. In one embodiment, the strongest peak is calculated as a combination of analyzing the overall peak height and area beneath the peak. For example, a moderately high but wide peak would be stronger than a very high peak that is extremely narrow. With the strongest putative peak identified, such as [0477] putative peak 225, a Gaussian 228 curve is fit to the peak 225. Once the Gaussian is fit, the width (W) of the Gaussian is determined and will be used as the peak width for future calculations.
  • As generally addressed above, the denoised, shifted, and baseline-corrected signal is not sufficiently processed for confidently calling the DNA composition of the biological sample. For example, although the baseline has generally been removed, there are still residual baseline effects present. These residual baseline effects are therefore removed to increase the accuracy and confidence in making identifications. [0478]
  • To remove the residual baseline effects, FIG. 45 shows that the [0479] putative peaks 218, 222, and 224 are removed from the baseline corrected signal. The peaks are removed by identifying a center line 230, 232, and 234 of the putative peaks 218, 222, and 224, respectively and removing an area to the left and to the right of the identified center line. For each putative peak, an area equal to twice the width (W) of the Gaussian is removed from the left of the center line, while an area equivalent to 50 daltons is removed from the right of the center line. It has been found that the area representing 50 daltons is adequate to sufficiently remove the effect of salt adducts which can be associated with an actual peak. Such adducts appear to the right of an actual peak and are a natural effect from the chemistry involved in acquiring a mass spectrum. Although a 50 Dalton buffer has been selected, it will be appreciated that other ranges or methods can be used to reduce or eliminate adduct effects.
  • The peaks are removed and remaining [0480] minima 247 located as shown in FIG. 46 with the minima 247 connected to create signal 245. A quartic polynomial is applied to signal 245 to generate a residual baseline 250 as shown in FIG. 47. The residual baseline 250 is subtracted from the signal 225 to generate the final signal 255 as indicated in FIG. 48. Although the residual baseline is the result of a quartic fit to signal 245, it will be appreciated that other techniques can be used to smooth or fit the residual baseline.
  • To determine peak height, as shown in FIG. 49, a Gaussian such as [0481] Gaussian 266, 268, and 270 is fit to each of the peaks, such as peaks 260, 262, and 264, respectively. Accordingly, the height of the Gaussian is determined as height 272, 274, and 276. Once the height of each Gaussian peak is determined, then the method of identifying a biological compound 35 can move into the genotyping phase 65 as shown in FIG. 25.
  • An indication of the confidence that each putative peak is an actual peak can be discerned by calculating a signal-to-noise ratio for each putative peak. Accordingly, putative peaks with a strong signal-to-noise ratio are generally more likely to be an actual peak than a putative peak with a lower signal-to-noise ratio. As described above and shown in FIG. 50, the height of each peak, such as [0482] height 272, 274, and 276, is determined for each peak, with the height being an indicator of signal strength for each peak. The noise profile, such as noise profile 97, is extrapolated into noise profile 280 across the identified peaks. At the center line of each of the peaks, a noise value is determined, such as noise value 282, 283, and 284. With a signal values and a noise values generated, signal-to-noise ratios can be calculated for each peak. For example, the signal-to-noise ratio for the first peak in FIG. 50 would be calculated as signal value 272 divided by noise value 282, and in a similar manner the signal-to-noise ratio of the middle peak in FIG. 50 would be determined as signal 274 divided by noise value 283.
  • Although the signal-to-noise ratio is generally a useful indicator of the presence of an actual peak, further processing has been found to increase the confidence by which a sample can be identified. For example, the signal-to-noise ratio for each peak in the exemplarly embodiment can be adjusted by the goodness of fit between a Gaussian and each putative peak. It is a characteristic of a mass spectrometer that sample material is detected in a manner that generally complies with a normal distribution. Accordingly, greater confidence will be associated with a putative signal having a Gaussian shape than a signal that has a less normal distribution. The error resulting from having a non-Gaussian shape can be referred to as a “residual error”. [0483]
  • Referring to FIG. 51, a residual error is calculated by taking a root mean square calculation between the Gaussian 293 and the [0484] putative peak 290 in the data signal. The calculation is performed on data within one width on either side of a center line of the Gaussian. The residual error is calculated as:
  • {square root}[(G−R)2/N],
  • where G is the Gaussian signal value, R is the putative peak value, and N is the number of points from −W to +W. The calculated residual error is used to generate an adjusted signal-to-noise ratio, as described below. [0485]
  • An adjusted signal noise ratio is calculated for each putative peak using the formula (S/N) * EXP[0486] (−1·R), where S/N is the signal-to-noise ratio, and R is the residual error determined above. Although the exemplary embodiment calculates an adjusted signal-to-noise ratio using a residual error for each peak, it will be appreciated that other techniques can be used to account for the goodness of fit between the Gaussian and the actual signal.
  • Referring now to FIG. 52, a probability is determined that a putative peak is an actual peak. In making the determination of peak probability, a [0487] probability profile 300 is generated where the adjusted signal-to-noise ratio is the x-axis and the probability is the y-axis. Probability is necessarily in the range between a 0% probability and a 100% probability, which is indicated as 1. Generally, the higher the adjusted signal-to-noise ratio, the greater the confidence that a putative peak is an actual peak.
  • At some target value for the adjusted signal-to-noise, it has been found that the probability is 100% that the putative peak is an actual peak and can confidently be used to identify the DNA composition of a biological sample. The target value of adjusted signal-to-noise ratio where the probability is assumed to be 100% is a variable parameter which is to be set according to application specific criteria. For example, the target signal-to-noise ratio will be adjusted depending upon trial experience, sample characteristics, and the acceptable error tolerance in the overall system. More specifically, for situations requiring a conservative approach where error cannot be tolerated, the target adjusted signal-to-noise ratio can be set to, for example, 10 and higher. Accordingly, 100% probability will not be assigned to a peak unless the adjusted signal-to-noise ratio is 10 or over. [0488]
  • In other situations, a more aggressive approach can be taken as sample data is more pronounced or the risk of error can be reduced. In such a situation, the system can be set to assume a 100% probability with a 5 or greater target signal-to-noise ratio. Of course, an intermediate signal-to-noise ratio target figure can be selected, such as 7, when a moderate risk of error can be assumed. Once the target adjusted signal-to-noise ratio is set for the method, then for any adjusted signal-to-noise ratio a probability can be determined that a putative peak is an actual peak. [0489]
  • Due to the chemistry involved in performing an identification test, especially a mass spectrometry test of a sample prepared by DNA amplifications, the allelic ratio between the signal strength of the highest peak and the signal strength of the second (or third and so on) highest peak should fall within an expected ratio. If the allelic ratio falls outside of normal guidelines, the exemplary embodiment imposes an allelic ratio penalty to the probability. For example, FIG. 53 shows an [0490] allelic penalty 315 which has an x-axis 317 that is the ratio between the signal strength of the second highest peak divided by signal strength of the highest peak. The yaxis 319 assigns a penalty between 0 and 1 depending on the determined allelic ratio. In the exemplary embodiment, it is assumed that allelic ratios over 30% are within the expected range and therefore no penalty is applied. Between a ratio of 10% and 30%, the penalty is linearly increased until at allelic ratios below 10% it is assumed the second-highest peak is not real. For allelic ratios between 10% and 30%, the allelic penalty chart 315 is used to determine a penalty 319, which is multiplied by the peak probability determined in FIG. 52 to determine a final peak probability. Although the exemplary embodiment incorporates an allelic ratio penalty to account for a possible chemistry error, it will be appreciated that other techniques can be used. Similar treatment will be applied to the other peaks.
  • With the peak probability of each peak determined, the statistical probability for various composition components can be determined, as an example, in order to determine the probability of each of three possible combinations of two peaks,—peak G, peak C and combinations GG, CC and GC. FIG. 54 shows an example where a most [0491] probable peak 325 is determined to have a final peak probability of 90%. Peak 325 is positioned such that it represents a G component in the biological sample. Accordingly, it can be maintained that there is a 90% probability that G exists in the biological sample. Also in the example shown in FIG. 54, the second highest probability is peak 330 which has a peak probability of 20%. Peak 330 is at a position associated with a C composition. Accordingly, it can be maintained that there is a 20% probability that C exists in the biological sample.
  • With the probability of G existing (90%) and the probability of C existing (20%) as a starting point, the probability of combinations of G and C existing can be calculated. For example, FIG. 54 indicates that the probability of GG existing 329 is calculated as 72%. This is calculated as the probability of GG is equal to the probability of G existing (90%) multiplied by the probability of C not existing (100% −20%). So if the probability of G existing is 90% and the probability of C not existing is 80%, the probability of GG is 72%. [0492]
  • In a similar manner, the probability of CC existing is equivalent to the probability of C existing (20%) multiplied by the probability of G not existing (100% −90%). As shown in FIG. 54, the probability of C existing is 20% while the probability of G not existing is 10%, so therefore the probability of CC is only 2%. Finally, the probability of GC existing is equal to the probability of G existing (90%) multiplied by the probability of C existing (20%). So if the probability of G existing is 90% and the probability of C existing is 20%, the probability of GC existing is 18%. In summary form, then, the probability of the composition of the biological sample is: [0493]
    probability of GG: 72%;
    probability of GC:    18%; and
    probability of CC:  2%.
  • Once the probabilities of each of the possible combinations has been determined, FIG. 55 is used to decide whether or not sufficient confidence exists to call the genotype. FIG. 55 shows a [0494] call chart 335 which has an x-axis 337 which is the ratio of the highest combination probability to the second highest combination probability. The yaxis 339 simply indicates whether the ratio is sufficiently high to justify calling the genotype. The value of the ratio can be indicated by M 340. The value of M is set depending upon trial data, sample composition, and the ability to accept error. For example, the value M can be set relatively high, such as to a value 4 so that the highest probability must be at least four times greater than the second highest probability before confidence is established to call a genotype. If a certain level of error can be acceptable, the value of M can be set to a more aggressive value, such as to 3, so that the ratio between the highest and second highest probabilities needs to be only a ratio of 3 or higher. Of course, moderate value can be selected for M when a moderate risk can be accepted. Using the example of FIG. 54, where the probability of GG was 72% and the probability of GC was 18%, the ratio between 72% and 18% is 4.0, therefore, whether M is set to 3, 3.5, or 4, the system would call the genotype as GG. Although the exemplary embodiment uses a ratio between the two highest peak probabilities to determine if a genotype confidently can be called, it will be appreciated that other methods can be substituted. It will also be appreciated that the above techniques can be used for calculating probabilities and choosing genotypes (or more general DNA patterns) containing of combinations of more than two peaks.
  • Referring now to FIG. 56, a flow chart is shown generally defining the process of statistically calling genotype described above. In FIG. 56 [0495] block 402 shows that the height of each peak is determined and that in block 404 a noise profile is extrapolated for each peak. The signal is determined from the height of each peak in block 406 and the noise for each peak is determined using the noise profile in block 408. In block 410, the signal-to-noise ratio is calculated for each peak. To account for a non-Gaussian peak shape, a residual error is determined in block 412 and an adjusted signal-to-noise ratio is calculated in block 414. Block 416 shows that a probability profile is developed, with the probability of each peak existing found in block 418. An allelic penalty can be applied in block 420, with the allelic penalty applied to the adjusted peak probability in block 422. The probability of each combination of components is calculated in block 424 with the ratio between the two highest probabilities being determined in block 426. If the ratio of probabilities exceeds a threshold value then the genotype is called in block 428.
  • In another embodiment, the computing device 20 (FIG. 24) supports “standardless” genotyping by identifying data peaks that contain putative SNPs. Standardless genotyping is used, for example, where insufficient information is known about the samples to determine a distribution of expected peak locations, against which an allelic penalty as described above can be reliably calculated. This permits the computing device to be used for identification of peaks that contain putative SNPs from data generated by any assay that fragments a targeted DNA molecule. For such standardless genotyping, peaks that are associated with an area under the data curve that deviates significantly from the typical area of other peaks in the data spectrum are identified and their corresponding mass (location along the x-axis) is determined. [0496]
  • More particularly, peaks that deviate significantly from the average area of other peaks in the data are identified, and the expected allelic ratio between data peaks is defined in terms of the ratio of the area under the data peaks. Theoretically, where each genetic loci has the same molar concentration of analyte, the area under each corresponding peak should be the same, thus producing a 1.0 ratio of the peak area between any two peaks. In accordance with the methods provided herein, peaks having a smaller ratio relative to the other peaks in the data will not be recognized as peaks. More particularly, peaks having an area ratio smaller than 30% relative to a nominal value for peak area will be assigned an allelic penalty. The mass of the remaining peaks (their location along the x-axis of the data) will be determined based on oligonucleotide standards. [0497]
  • FIG. 57 shows a flow diagram representation of the processing by the computing device 20 (FIG. 24) when performing standardless genotyping. In the first operation, represented by the flow diagram box numbered 502, the computing device receives data from the mass spectrometer. Next, the height of each putative peak in the data sample is determined, as indicated by the [0498] block 504. After the height of each peak in the mass spectrometer data is determined, a de-noise process 505 is performed, beginning with an extrapolation of the noise profile (block 506), followed by finding the noise of each peak (block 508) and calculating the signal to noise ratio for each data sample (block 510). Each of these operations can be performed in accordance with the description above for denoise operations 45 of FIG. 25. Other suitable denoise operations will occur to those skilled in the art.
  • The next operation is to find the residual error associated with each data point. This is represented by the [0499] block 512 in FIG. 57. The next step, block 514, involves calculating an adjusted signal to noise ratio for each identified peak. A probability profile is developed next (block 516), followed by a determination of the peak probabilities at block 518. In an exemplary embodiment, the denoise operations of FIG. 57, comprising block 502 to block 518, comprise the corresponding operations described above in conjunction with FIG. 56 for block 402 through block 418, respectively.
  • The next action for the standardless genotype processing is to determine an allelic penalty for each peak, indicated by the [0500] block 524. As noted above, the standardless genotype processing of FIG. 57 determines an allelic penalty by comparing area under the peaks. Therefore, rather than compare signal strength ratios to determine an allelic penalty, such as described above for FIG. 53, the standardless processing determines the area under each of the identified peaks and compares the ratio of those areas. Determining the area under each peak can be computed using conventional numerical analysis techniques for calculating the area under a curve for experimental data.
  • Thus, the allelic penalty is assigned in accordance with FIG. 58, which shows that no penalty is assigned to peaks having a peak area relative to an expected average area value that is greater than 0.30 (30%). The allelic penalty is applied to the peak probability value, which can be determined according to the process such as described in FIG. 52. It should be apparent from FIG. 58 that the allelic penalty imposed for peaks below a ratio of 30% is that such peaks will be removed from further measurement and processing. Other penalty schemes, however, can be imposed in accordance with knowledge about the data being processed, as determined by those skilled in the art. [0501]
  • After the allelic penalty has been determined and applied, the standardless genotype processing compares the location of the remaining putative peaks to oligonucleotide standards to determine corresponding masses in the processing for [0502] block 524. For standardless genotype data, the processing of the block 524 is performed to determine mass and genotype, rather than performing the operations corresponding to block 424, 426, and 428 of FIG. 33. Techniques for performing such comparisons and determining mass will be known to those skilled in the art.
  • In another embodiment, the computing device 20 (FIG. 24) permits the detection and determination of the mass (location along the x-axis of the data) of the sense and antisense strand of fragments generated in the assay. If desired, the computing device can also detect and determine the quantity (area under each peak) of the respective sense and antisense strands, using a similar technique to that described above for standardless genotype processing. The data generated for each type of strand can then be combined to achieve a data redundancy and to thereby increase the confidence level of the determined genotype. This technique obviates primer peaks that are often observed in data from other diagnostic methods, thereby permitting a higher level of multiplexing. In addition, when quantitation is used in pooling experiments, the ratio of the measured peak areas is more reliably calculated than the peak identifying technique, due to data redundancy. [0503]
  • FIG. 23 is a flow diagram that illustrates the processing implemented by the [0504] computing device 20 to perform sense and antisense processing. In the first operation, represented by the flow diagram box numbered 602, the computing device receives data from the mass spectrometer. This data will include data for the sense strand and antisense strand of assay fragments. Next, the height of each putative peak in the data sample is determined, as indicated by the block 604. After the height of each peak in the mass spectrometer data is determined, a de-noise process 605 is performed, beginning with an operation that extrapolates the noise profile (block 606), followed by finding the noise of each peak (block 608) and calculating the signal to noise ratio for each data sample (block 610). Each of these operations can be performed in accordance with the description above for the denoise operations 45 of FIG. 25. Other suitable denoise operations will occur to those skilled in the art. The next operation is to find the residual error associated with each data point. This is represented by the block 612 in FIG. 36.
  • After the residual error for the data of the sense strand and antisense strand has been performed, processing to identify the genotypes will be performed for the sense strand and also for the antisense strand. Therefore, FIG. 23 shows that processing includes sense strand processing (block 630) and antisense strand processing (block 640). Each [0505] block 630, 640 includes processing that corresponds to adjusting the signal to noise ratio, developing a probability profile, determining an allelic penalty, adjusting the peak probability by the allelic penalty, calculating genotype probabilities, and testing genotype probability ratios, such as described above in conjunction with blocks 414 through 426 of FIG. 56. The processing of each block 630, 640 can, if desired, include standardless processing operations such as described above in conjunction with FIG. 57. The standardless processing can be included in place of or in addition to the processing operations of FIG. 56.
  • After the genotype probability processing is completed, the data from the sense strand and antisense strand processing is combined and compared to expected database values to obtain the benefits of data redundancy as between the sense strand and antisense strand. Those skilled in the art will understand techniques to take advantage of known data redundancies between a sense strand and antisense strand of assay fragments. This processing is represented by the [0506] block 650. After the data from the two strands is combined for processing, the genotype processing is performed (block 660) and the genotype is identified.
  • Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims. [0507]
  • 0
    SEQUENCE LISTING
    <160> NUMBER OF SEQ ID NOS: 118
    <210> SEQ ID NO 1
    <211> LENGTH: 361
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <400> SEQUENCE: 1
    ctgaggacct ggtcctctga ctgctctttt cacccatcta cagtccccct tgccgtccca 60
    agcaatggat gatttgatgc tgtccccgga cgatattgaa caatggttca ctgaagaccc 120
    aggtccagat gaagctccca gaatgccaga ggctgctccc cgcgtggccc ctgcaccagc 180
    agctcctaca ccggcggccc ctgcaccagc cccctcctgg cccctgtcat cttctgtccc 240
    ttcccagaaa acctaccagg gcagctacgg tttccgtctg ggcttcttgc attctgggac 300
    agccaagtct gtgacttgca cggtcagttg ccctgagggg ctggcttcca tgagacttca 360
    a 361
    <210> SEQ ID NO 2
    <211> LENGTH: 44
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 2
    cccagtcacg acgttgtaaa acgctgagga cctggtcctc tgac 44
    <210> SEQ ID NO 3
    <211> LENGTH: 42
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 3
    agcggataac aatttcacac aggttgaagt ctcatggaag cc 42
    <210> SEQ ID NO 4
    <211> LENGTH: 17
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 4
    gccagaggct gctcccc 17
    <210> SEQ ID NO 5
    <211> LENGTH: 17
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 5
    gccagaggct gctcccc 17
    <210> SEQ ID NO 6
    <211> LENGTH: 19
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 6
    gccagaggct gctccccgc 19
    <210> SEQ ID NO 7
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 7
    gccagaggct gctccccc 18
    <210> SEQ ID NO 8
    <211> LENGTH: 161
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <400> SEQUENCE: 8
    gtccgtcaga acccatgcgg cagcaaggcc tgccgccgcc tcttcggccc agtggacagc 60
    gagcagctga gccgcgactg tgatgcgcta atggcgggct gcatccagga ggcccgtgag 120
    cgatggaact tcgactttgt caccgagaca ccactggagg g 161
    <210> SEQ ID NO 9
    <211> LENGTH: 43
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 9
    cccagtcacg acgttgtaaa acggtccgtc agaacccatg cgg 43
    <210> SEQ ID NO 10
    <211> LENGTH: 44
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 10
    agcggataac aatttcacac aggctccagt ggtgtctcgg tgac 44
    <210> SEQ ID NO 11
    <211> LENGTH: 15
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 11
    cagcgagcag ctgag 15
    <210> SEQ ID NO 12
    <211> LENGTH: 15
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 12
    cagcgagcag ctgag 15
    <210> SEQ ID NO 13
    <211> LENGTH: 16
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 13
    cagcgagcag ctgagc 16
    <210> SEQ ID NO 14
    <211> LENGTH: 17
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 14
    cagcgagcag ctgagac 17
    <210> SEQ ID NO 15
    <211> LENGTH: 205
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <400> SEQUENCE: 15
    gcgctccatt catctcttca tcgactctct gttgaatgaa gaaaatccaa gtaaggccta 60
    caggtgcagt tccaaggaag cctttgagaa agggctctgc ttgagttgta gaaagaaccg 120
    ctgcaacaat ctgggctatg agatcaataa agtcagagcc aaaagaagca gcaaaatgta 180
    cctgaagact cgttctcaga tgccc 205
    <210> SEQ ID NO 16
    <211> LENGTH: 42
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primers
    <400> SEQUENCE: 16
    cccagtcacg acgttgtaaa acggcgctcc attcatctct tc 42
    <210> SEQ ID NO 17
    <211> LENGTH: 42
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 17
    agcggataac aatttcacac agggggcatc tgagaacgag tc 42
    <210> SEQ ID NO 18
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 18
    caatctgggc tatgagatca 20
    <210> SEQ ID NO 19
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 19
    caatctgggc tatgagatca 20
    <210> SEQ ID NO 20
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 20
    caatctgggc tatgagatca a 21
    <210> SEQ ID NO 21
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 21
    caatctgggc tatgagatca gt 22
    <210> SEQ ID NO 22
    <211> LENGTH: 60
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 22
    gtgccggcta ctcggatggc agcaaggact cctgcaaggg ggacagtgga ggcccacatg 60
    <210> SEQ ID NO 23
    <211> LENGTH: 60
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 23
    ccacccacta ccggggcacg tggtacctga cgggcatcgt cagctggggc cagggctgcg 60
    <210> SEQ ID NO 24
    <211> LENGTH: 42
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 24
    cccagtcacg acgttgtaaa acgatggcag caaggactcc tg 42
    <210> SEQ ID NO 25
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 25
    cacatgccac ccactacc 18
    <210> SEQ ID NO 26
    <211> LENGTH: 43
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 26
    agcggataac aatttcacac aggtgacgat gcccgtcagg tac 43
    <210> SEQ ID NO 27
    <211> LENGTH: 15
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 27
    atgccaccca ctacc 15
    <210> SEQ ID NO 28
    <211> LENGTH: 19
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 28
    cacatgccac ccactaccg 19
    <210> SEQ ID NO 29
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 29
    cacatgccac ccactaccag 20
    <210> SEQ ID NO 30
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Probe
    <400> SEQUENCE: 30
    agcggataac aatttcacac agg 23
    <210> SEQ ID NO 31
    <211> LENGTH: 2363
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (138)...(2126)
    <223> OTHER INFORMATION: AKAP-10
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: GenBank AF037439
    <309> DATABASE ENTRY DATE: 1997-12-21
    <400> SEQUENCE: 31
    gcggcttgtt gataatatgg cggctggagc tgcctgggca tcccgaggag gcggtggggc 60
    ccactcccgg aagaagggtc ccttttcgcg ctagtgcagc ggcccctctg gacccggaag 120
    tccgggccgg ttgctga atg agg gga gcc ggg ccc tcc ccg cgc cag tcc 170
    Met Arg Gly Ala Gly Pro Ser Pro Arg Gln Ser
    1 5 10
    ccc cgc acc ctc cgt ccc gac ccg ggc ccc gcc atg tcc ttc ttc cgg 218
    Pro Arg Thr Leu Arg Pro Asp Pro Gly Pro Ala Met Ser Phe Phe Arg
    15 20 25
    cgg aaa gtg aaa ggc aaa gaa caa gag aag acc tca gat gtg aag tcc 266
    Arg Lys Val Lys Gly Lys Glu Gln Glu Lys Thr Ser Asp Val Lys Ser
    30 35 40
    att aaa gct tca ata tcc gta cat tcc cca caa aaa agc act aaa aat 314
    Ile Lys Ala Ser Ile Ser Val His Ser Pro Gln Lys Ser Thr Lys Asn
    45 50 55
    cat gcc ttg ctg gag gct gca gga cca agt cat gtt gca atc aat gcc 362
    His Ala Leu Leu Glu Ala Ala Gly Pro Ser His Val Ala Ile Asn Ala
    60 65 70 75
    att tct gcc aac atg gac tcc ttt tca agt agc agg aca gcc aca ctt 410
    Ile Ser Ala Asn Met Asp Ser Phe Ser Ser Ser Arg Thr Ala Thr Leu
    80 85 90
    aag aag cag cca agc cac atg gag gct gct cat ttt ggt gac ctg ggc 458
    Lys Lys Gln Pro Ser His Met Glu Ala Ala His Phe Gly Asp Leu Gly
    95 100 105
    aga tct tgt ctg gac tac cag act caa gag acc aaa tca agc ctt tct 506
    Arg Ser Cys Leu Asp Tyr Gln Thr Gln Glu Thr Lys Ser Ser Leu Ser
    110 115 120
    aag acc ctt gaa caa gtc ttg cac gac act att gtc ctc cct tac ttc 554
    Lys Thr Leu Glu Gln Val Leu His Asp Thr Ile Val Leu Pro Tyr Phe
    125 130 135
    att caa ttc atg gaa ctt cgg cga atg gag cat ttg gtg aaa ttt tgg 602
    Ile Gln Phe Met Glu Leu Arg Arg Met Glu His Leu Val Lys Phe Trp
    140 145 150 155
    tta gag gct gaa agt ttt cat tca aca act tgg tcg cga ata aga gca 650
    Leu Glu Ala Glu Ser Phe His Ser Thr Thr Trp Ser Arg Ile Arg Ala
    160 165 170
    cac agt cta aac aca atg aag cag agc tca ctg gct gag cct gtc tct 698
    His Ser Leu Asn Thr Met Lys Gln Ser Ser Leu Ala Glu Pro Val Ser
    175 180 185
    cca tct aaa aag cat gaa act aca gcg tct ttt tta act gat tct ctt 746
    Pro Ser Lys Lys His Glu Thr Thr Ala Ser Phe Leu Thr Asp Ser Leu
    190 195 200
    gat aag aga ttg gag gat tct ggc tca gca cag ttg ttt atg act cat 794
    Asp Lys Arg Leu Glu Asp Ser Gly Ser Ala Gln Leu Phe Met Thr His
    205 210 215
    tca gaa gga att gac ctg aat aat aga act aac agc act cag aat cac 842
    Ser Glu Gly Ile Asp Leu Asn Asn Arg Thr Asn Ser Thr Gln Asn His
    220 225 230 235
    ttg ctg ctt tcc cag gaa tgt gac agt gcc cat tct ctc cgt ctt gaa 890
    Leu Leu Leu Ser Gln Glu Cys Asp Ser Ala His Ser Leu Arg Leu Glu
    240 245 250
    atg gcc aga gca gga act cac caa gtt tcc atg gaa acc caa gaa tct 938
    Met Ala Arg Ala Gly Thr His Gln Val Ser Met Glu Thr Gln Glu Ser
    255 260 265
    tcc tct aca ctt aca gta gcc agt aga aat agt ccc gct tct cca cta 986
    Ser Ser Thr Leu Thr Val Ala Ser Arg Asn Ser Pro Ala Ser Pro Leu
    270 275 280
    aaa gaa ttg tca gga aaa cta atg aaa agt ata gaa caa gat gca gtg 1034
    Lys Glu Leu Ser Gly Lys Leu Met Lys Ser Ile Glu Gln Asp Ala Val
    285 290 295
    aat act ttt acc aaa tat ata tct cca gat gct gct aaa cca ata cca 1082
    Asn Thr Phe Thr Lys Tyr Ile Ser Pro Asp Ala Ala Lys Pro Ile Pro
    300 305 310 315
    att aca gaa gca atg aga aat gac atc ata gca agg att tgt gga gaa 1130
    Ile Thr Glu Ala Met Arg Asn Asp Ile Ile Ala Arg Ile Cys Gly Glu
    320 325 330
    gat gga cag gtg gat ccc aac tgt ttc gtt ttg gca cag tcc ata gtc 1178
    Asp Gly Gln Val Asp Pro Asn Cys Phe Val Leu Ala Gln Ser Ile Val
    335 340 345
    ttt agt gca atg gag caa gag cac ttt agt gag ttt ctg cga agt cac 1226
    Phe Ser Ala Met Glu Gln Glu His Phe Ser Glu Phe Leu Arg Ser His
    350 355 360
    cat ttc tgt aaa tac cag att gaa gtg ctg acc agt gga act gtt tac 1274
    His Phe Cys Lys Tyr Gln Ile Glu Val Leu Thr Ser Gly Thr Val Tyr
    365 370 375
    ctg gct gac att ctc ttc tgt gag tca gcc ctc ttt tat ttc tct gag 1322
    Leu Ala Asp Ile Leu Phe Cys Glu Ser Ala Leu Phe Tyr Phe Ser Glu
    380 385 390 395
    tac atg gaa aaa gag gat gca gtg aat atc tta caa ttc tgg ttg gca 1370
    Tyr Met Glu Lys Glu Asp Ala Val Asn Ile Leu Gln Phe Trp Leu Ala
    400 405 410
    gca gat aac ttc cag tct cag ctt gct gcc aaa aag ggg caa tat gat 1418
    Ala Asp Asn Phe Gln Ser Gln Leu Ala Ala Lys Lys Gly Gln Tyr Asp
    415 420 425
    gga cag gag gca cag aat gat gcc atg att tta tat gac aag tac ttc 1466
    Gly Gln Glu Ala Gln Asn Asp Ala Met Ile Leu Tyr Asp Lys Tyr Phe
    430 435 440
    tcc ctc caa gcc aca cat cct ctt gga ttt gat gat gtt gta cga tta 1514
    Ser Leu Gln Ala Thr His Pro Leu Gly Phe Asp Asp Val Val Arg Leu
    445 450 455
    gaa att gaa tcc aat atc tgc agg gaa ggt ggg cca ctc ccc aac tgt 1562
    Glu Ile Glu Ser Asn Ile Cys Arg Glu Gly Gly Pro Leu Pro Asn Cys
    460 465 470 475
    ttc aca act cca tta cgt cag gcc tgg aca acc atg gag aag gtc ttt 1610
    Phe Thr Thr Pro Leu Arg Gln Ala Trp Thr Thr Met Glu Lys Val Phe
    480 485 490
    ttg cct ggc ttt ctg tcc agc aat ctt tat tat aaa tat ttg aat gat 1658
    Leu Pro Gly Phe Leu Ser Ser Asn Leu Tyr Tyr Lys Tyr Leu Asn Asp
    495 500 505
    ctc atc cat tcg gtt cga gga gat gaa ttt ctg ggc ggg aac gtg tcg 1706
    Leu Ile His Ser Val Arg Gly Asp Glu Phe Leu Gly Gly Asn Val Ser
    510 515 520
    ccg act gct cct ggc tct gtt ggc cct cct gat gag tct cac cca ggg 1754
    Pro Thr Ala Pro Gly Ser Val Gly Pro Pro Asp Glu Ser His Pro Gly
    525 530 535
    agt tct gac agc tct gcg tct cag tcc agt gtg aaa aaa gcc agt att 1802
    Ser Ser Asp Ser Ser Ala Ser Gln Ser Ser Val Lys Lys Ala Ser Ile
    540 545 550 555
    aaa ata ctg aaa aat ttt gat gaa gcg ata att gtg gat gcg gca agt 1850
    Lys Ile Leu Lys Asn Phe Asp Glu Ala Ile Ile Val Asp Ala Ala Ser
    560 565 570
    ctg gat cca gaa tct tta tat caa cgg aca tat gcc ggg aag atg aca 1898
    Leu Asp Pro Glu Ser Leu Tyr Gln Arg Thr Tyr Ala Gly Lys Met Thr
    575 580 585
    ttt gga aga gtg agt gac ttg ggg caa ttc atc cgg gaa tct gag cct 1946
    Phe Gly Arg Val Ser Asp Leu Gly Gln Phe Ile Arg Glu Ser Glu Pro
    590 595 600
    gaa cct gat gta agg aaa tca aaa gga tcc atg ttc tca caa gct atg 1994
    Glu Pro Asp Val Arg Lys Ser Lys Gly Ser Met Phe Ser Gln Ala Met
    605 610 615
    aag aaa tgg gtg caa gga aat act gat gag gcc cag gaa gag cta gct 2042
    Lys Lys Trp Val Gln Gly Asn Thr Asp Glu Ala Gln Glu Glu Leu Ala
    620 625 630 635
    tgg aag att gct aaa atg ata gtc agt gac att atg cag cag gct cag 2090
    Trp Lys Ile Ala Lys Met Ile Val Ser Asp Ile Met Gln Gln Ala Gln
    640 645 650
    tat gat caa ccg tta gag aaa tct aca aag tta tga ctcaaaactt 2136
    Tyr Asp Gln Pro Leu Glu Lys Ser Thr Lys Leu *
    655 660
    gagataaagg aaatctgctt gtgaaaaata agagaacttt tttcccttgg ttggattctt 2196
    caacacagcc aatgaaaaca gcactatatt tctgatctgt cactgttgtt tccagggaga 2256
    gaatggggag acaatcctag gacttccacc ctaatgcagt tacctgtagg gcataattgg 2316
    atggcacatg atgtttcaca cagtgaggag tctttaaagg ttaccaa 2363
    <210> SEQ ID NO 32
    <211> LENGTH: 662
    <212> TYPE: PRT
    <213> ORGANISM: Homo Sapien
    <400> SEQUENCE: 32
    Met Arg Gly Ala Gly Pro Ser Pro Arg Gln Ser Pro Arg Thr Leu Arg
    1 5 10 15
    Pro Asp Pro Gly Pro Ala Met Ser Phe Phe Arg Arg Lys Val Lys Gly
    20 25 30
    Lys Glu Gln Glu Lys Thr Ser Asp Val Lys Ser Ile Lys Ala Ser Ile
    35 40 45
    Ser Val His Ser Pro Gln Lys Ser Thr Lys Asn His Ala Leu Leu Glu
    50 55 60
    Ala Ala Gly Pro Ser His Val Ala Ile Asn Ala Ile Ser Ala Asn Met
    65 70 75 80
    Asp Ser Phe Ser Ser Ser Arg Thr Ala Thr Leu Lys Lys Gln Pro Ser
    85 90 95
    His Met Glu Ala Ala His Phe Gly Asp Leu Gly Arg Ser Cys Leu Asp
    100 105 110
    Tyr Gln Thr Gln Glu Thr Lys Ser Ser Leu Ser Lys Thr Leu Glu Gln
    115 120 125
    Val Leu His Asp Thr Ile Val Leu Pro Tyr Phe Ile Gln Phe Met Glu
    130 135 140
    Leu Arg Arg Met Glu His Leu Val Lys Phe Trp Leu Glu Ala Glu Ser
    145 150 155 160
    Phe His Ser Thr Thr Trp Ser Arg Ile Arg Ala His Ser Leu Asn Thr
    165 170 175
    Met Lys Gln Ser Ser Leu Ala Glu Pro Val Ser Pro Ser Lys Lys His
    180 185 190
    Glu Thr Thr Ala Ser Phe Leu Thr Asp Ser Leu Asp Lys Arg Leu Glu
    195 200 205
    Asp Ser Gly Ser Ala Gln Leu Phe Met Thr His Ser Glu Gly Ile Asp
    210 215 220
    Leu Asn Asn Arg Thr Asn Ser Thr Gln Asn His Leu Leu Leu Ser Gln
    225 230 235 240
    Glu Cys Asp Ser Ala His Ser Leu Arg Leu Glu Met Ala Arg Ala Gly
    245 250 255
    Thr His Gln Val Ser Met Glu Thr Gln Glu Ser Ser Ser Thr Leu Thr
    260 265 270
    Val Ala Ser Arg Asn Ser Pro Ala Ser Pro Leu Lys Glu Leu Ser Gly
    275 280 285
    Lys Leu Met Lys Ser Ile Glu Gln Asp Ala Val Asn Thr Phe Thr Lys
    290 295 300
    Tyr Ile Ser Pro Asp Ala Ala Lys Pro Ile Pro Ile Thr Glu Ala Met
    305 310 315 320
    Arg Asn Asp Ile Ile Ala Arg Ile Cys Gly Glu Asp Gly Gln Val Asp
    325 330 335
    Pro Asn Cys Phe Val Leu Ala Gln Ser Ile Val Phe Ser Ala Met Glu
    340 345 350
    Gln Glu His Phe Ser Glu Phe Leu Arg Ser His His Phe Cys Lys Tyr
    355 360 365
    Gln Ile Glu Val Leu Thr Ser Gly Thr Val Tyr Leu Ala Asp Ile Leu
    370 375 380
    Phe Cys Glu Ser Ala Leu Phe Tyr Phe Ser Glu Tyr Met Glu Lys Glu
    385 390 395 400
    Asp Ala Val Asn Ile Leu Gln Phe Trp Leu Ala Ala Asp Asn Phe Gln
    405 410 415
    Ser Gln Leu Ala Ala Lys Lys Gly Gln Tyr Asp Gly Gln Glu Ala Gln
    420 425 430
    Asn Asp Ala Met Ile Leu Tyr Asp Lys Tyr Phe Ser Leu Gln Ala Thr
    435 440 445
    His Pro Leu Gly Phe Asp Asp Val Val Arg Leu Glu Ile Glu Ser Asn
    450 455 460
    Ile Cys Arg Glu Gly Gly Pro Leu Pro Asn Cys Phe Thr Thr Pro Leu
    465 470 475 480
    Arg Gln Ala Trp Thr Thr Met Glu Lys Val Phe Leu Pro Gly Phe Leu
    485 490 495
    Ser Ser Asn Leu Tyr Tyr Lys Tyr Leu Asn Asp Leu Ile His Ser Val
    500 505 510
    Arg Gly Asp Glu Phe Leu Gly Gly Asn Val Ser Pro Thr Ala Pro Gly
    515 520 525
    Ser Val Gly Pro Pro Asp Glu Ser His Pro Gly Ser Ser Asp Ser Ser
    530 535 540
    Ala Ser Gln Ser Ser Val Lys Lys Ala Ser Ile Lys Ile Leu Lys Asn
    545 550 555 560
    Phe Asp Glu Ala Ile Ile Val Asp Ala Ala Ser Leu Asp Pro Glu Ser
    565 570 575
    Leu Tyr Gln Arg Thr Tyr Ala Gly Lys Met Thr Phe Gly Arg Val Ser
    580 585 590
    Asp Leu Gly Gln Phe Ile Arg Glu Ser Glu Pro Glu Pro Asp Val Arg
    595 600 605
    Lys Ser Lys Gly Ser Met Phe Ser Gln Ala Met Lys Lys Trp Val Gln
    610 615 620
    Gly Asn Thr Asp Glu Ala Gln Glu Glu Leu Ala Trp Lys Ile Ala Lys
    625 630 635 640
    Met Ile Val Ser Asp Ile Met Gln Gln Ala Gln Tyr Asp Gln Pro Leu
    645 650 655
    Glu Lys Ser Thr Lys Leu
    660
    <210> SEQ ID NO 33
    <211> LENGTH: 2363
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (138)...(2126)
    <223> OTHER INFORMATION: AKAP-10-5
    <220> FEATURE:
    <221> NAME/KEY: allele
    <222> LOCATION: 2073
    <223> OTHER INFORMATION: Single Nucleotide Polymorphism: A to G
    <400> SEQUENCE: 33
    gcggcttgtt gataatatgg cggctggagc tgcctgggca tcccgaggag gcggtggggc 60
    ccactcccgg aagaagggtc ccttttcgcg ctagtgcagc ggcccctctg gacccggaag 120
    tccgggccgg ttgctga atg agg gga gcc ggg ccc tcc ccg cgc cag tcc 170
    Met Arg Gly Ala Gly Pro Ser Pro Arg Gln Ser
    1 5 10
    ccc cgc acc ctc cgt ccc gac ccg ggc ccc gcc atg tcc ttc ttc cgg 218
    Pro Arg Thr Leu Arg Pro Asp Pro Gly Pro Ala Met Ser Phe Phe Arg
    15 20 25
    cgg aaa gtg aaa ggc aaa gaa caa gag aag acc tca gat gtg aag tcc 266
    Arg Lys Val Lys Gly Lys Glu Gln Glu Lys Thr Ser Asp Val Lys Ser
    30 35 40
    att aaa gct tca ata tcc gta cat tcc cca caa aaa agc act aaa aat 314
    Ile Lys Ala Ser Ile Ser Val His Ser Pro Gln Lys Ser Thr Lys Asn
    45 50 55
    cat gcc ttg ctg gag gct gca gga cca agt cat gtt gca atc aat gcc 362
    His Ala Leu Leu Glu Ala Ala Gly Pro Ser His Val Ala Ile Asn Ala
    60 65 70 75
    att tct gcc aac atg gac tcc ttt tca agt agc agg aca gcc aca ctt 410
    Ile Ser Ala Asn Met Asp Ser Phe Ser Ser Ser Arg Thr Ala Thr Leu
    80 85 90
    aag aag cag cca agc cac atg gag gct gct cat ttt ggt gac ctg ggc 458
    Lys Lys Gln Pro Ser His Met Glu Ala Ala His Phe Gly Asp Leu Gly
    95 100 105
    aga tct tgt ctg gac tac cag act caa gag acc aaa tca agc ctt tct 506
    Arg Ser Cys Leu Asp Tyr Gln Thr Gln Glu Thr Lys Ser Ser Leu Ser
    110 115 120
    aag acc ctt gaa caa gtc ttg cac gac act att gtc ctc cct tac ttc 554
    Lys Thr Leu Glu Gln Val Leu His Asp Thr Ile Val Leu Pro Tyr Phe
    125 130 135
    att caa ttc atg gaa ctt cgg cga atg gag cat ttg gtg aaa ttt tgg 602
    Ile Gln Phe Met Glu Leu Arg Arg Met Glu His Leu Val Lys Phe Trp
    140 145 150 155
    tta gag gct gaa agt ttt cat tca aca act tgg tcg cga ata aga gca 650
    Leu Glu Ala Glu Ser Phe His Ser Thr Thr Trp Ser Arg Ile Arg Ala
    160 165 170
    cac agt cta aac aca atg aag cag agc tca ctg gct gag cct gtc tct 698
    His Ser Leu Asn Thr Met Lys Gln Ser Ser Leu Ala Glu Pro Val Ser
    175 180 185
    cca tct aaa aag cat gaa act aca gcg tct ttt tta act gat tct ctt 746
    Pro Ser Lys Lys His Glu Thr Thr Ala Ser Phe Leu Thr Asp Ser Leu
    190 195 200
    gat aag aga ttg gag gat tct ggc tca gca cag ttg ttt atg act cat 794
    Asp Lys Arg Leu Glu Asp Ser Gly Ser Ala Gln Leu Phe Met Thr His
    205 210 215
    tca gaa gga att gac ctg aat aat aga act aac agc act cag aat cac 842
    Ser Glu Gly Ile Asp Leu Asn Asn Arg Thr Asn Ser Thr Gln Asn His
    220 225 230 235
    ttg ctg ctt tcc cag gaa tgt gac agt gcc cat tct ctc cgt ctt gaa 890
    Leu Leu Leu Ser Gln Glu Cys Asp Ser Ala His Ser Leu Arg Leu Glu
    240 245 250
    atg gcc aga gca gga act cac caa gtt tcc atg gaa acc caa gaa tct 938
    Met Ala Arg Ala Gly Thr His Gln Val Ser Met Glu Thr Gln Glu Ser
    255 260 265
    tcc tct aca ctt aca gta gcc agt aga aat agt ccc gct tct cca cta 986
    Ser Ser Thr Leu Thr Val Ala Ser Arg Asn Ser Pro Ala Ser Pro Leu
    270 275 280
    aaa gaa ttg tca gga aaa cta atg aaa agt ata gaa caa gat gca gtg 1034
    Lys Glu Leu Ser Gly Lys Leu Met Lys Ser Ile Glu Gln Asp Ala Val
    285 290 295
    aat act ttt acc aaa tat ata tct cca gat gct gct aaa cca ata cca 1082
    Asn Thr Phe Thr Lys Tyr Ile Ser Pro Asp Ala Ala Lys Pro Ile Pro
    300 305 310 315
    att aca gaa gca atg aga aat gac atc ata gca agg att tgt gga gaa 1130
    Ile Thr Glu Ala Met Arg Asn Asp Ile Ile Ala Arg Ile Cys Gly Glu
    320 325 330
    gat gga cag gtg gat ccc aac tgt ttc gtt ttg gca cag tcc ata gtc 1178
    Asp Gly Gln Val Asp Pro Asn Cys Phe Val Leu Ala Gln Ser Ile Val
    335 340 345
    ttt agt gca atg gag caa gag cac ttt agt gag ttt ctg cga agt cac 1226
    Phe Ser Ala Met Glu Gln Glu His Phe Ser Glu Phe Leu Arg Ser His
    350 355 360
    cat ttc tgt aaa tac cag att gaa gtg ctg acc agt gga act gtt tac 1274
    His Phe Cys Lys Tyr Gln Ile Glu Val Leu Thr Ser Gly Thr Val Tyr
    365 370 375
    ctg gct gac att ctc ttc tgt gag tca gcc ctc ttt tat ttc tct gag 1322
    Leu Ala Asp Ile Leu Phe Cys Glu Ser Ala Leu Phe Tyr Phe Ser Glu
    380 385 390 395
    tac atg gaa aaa gag gat gca gtg aat atc tta caa ttc tgg ttg gca 1370
    Tyr Met Glu Lys Glu Asp Ala Val Asn Ile Leu Gln Phe Trp Leu Ala
    400 405 410
    gca gat aac ttc cag tct cag ctt gct gcc aaa aag ggg caa tat gat 1418
    Ala Asp Asn Phe Gln Ser Gln Leu Ala Ala Lys Lys Gly Gln Tyr Asp
    415 420 425
    gga cag gag gca cag aat gat gcc atg att tta tat gac aag tac ttc 1466
    Gly Gln Glu Ala Gln Asn Asp Ala Met Ile Leu Tyr Asp Lys Tyr Phe
    430 435 440
    tcc ctc caa gcc aca cat cct ctt gga ttt gat gat gtt gta cga tta 1514
    Ser Leu Gln Ala Thr His Pro Leu Gly Phe Asp Asp Val Val Arg Leu
    445 450 455
    gaa att gaa tcc aat atc tgc agg gaa ggt ggg cca ctc ccc aac tgt 1562
    Glu Ile Glu Ser Asn Ile Cys Arg Glu Gly Gly Pro Leu Pro Asn Cys
    460 465 470 475
    ttc aca act cca tta cgt cag gcc tgg aca acc atg gag aag gtc ttt 1610
    Phe Thr Thr Pro Leu Arg Gln Ala Trp Thr Thr Met Glu Lys Val Phe
    480 485 490
    ttg cct ggc ttt ctg tcc agc aat ctt tat tat aaa tat ttg aat gat 1658
    Leu Pro Gly Phe Leu Ser Ser Asn Leu Tyr Tyr Lys Tyr Leu Asn Asp
    495 500 505
    ctc atc cat tcg gtt cga gga gat gaa ttt ctg ggc ggg aac gtg tcg 1706
    Leu Ile His Ser Val Arg Gly Asp Glu Phe Leu Gly Gly Asn Val Ser
    510 515 520
    ccg act gct cct ggc tct gtt ggc cct cct gat gag tct cac cca ggg 1754
    Pro Thr Ala Pro Gly Ser Val Gly Pro Pro Asp Glu Ser His Pro Gly
    525 530 535
    agt tct gac agc tct gcg tct cag tcc agt gtg aaa aaa gcc agt att 1802
    Ser Ser Asp Ser Ser Ala Ser Gln Ser Ser Val Lys Lys Ala Ser Ile
    540 545 550 555
    aaa ata ctg aaa aat ttt gat gaa gcg ata att gtg gat gcg gca agt 1850
    Lys Ile Leu Lys Asn Phe Asp Glu Ala Ile Ile Val Asp Ala Ala Ser
    560 565 570
    ctg gat cca gaa tct tta tat caa cgg aca tat gcc ggg aag atg aca 1898
    Leu Asp Pro Glu Ser Leu Tyr Gln Arg Thr Tyr Ala Gly Lys Met Thr
    575 580 585
    ttt gga aga gtg agt gac ttg ggg caa ttc atc cgg gaa tct gag cct 1946
    Phe Gly Arg Val Ser Asp Leu Gly Gln Phe Ile Arg Glu Ser Glu Pro
    590 595 600
    gaa cct gat gta agg aaa tca aaa gga tcc atg ttc tca caa gct atg 1994
    Glu Pro Asp Val Arg Lys Ser Lys Gly Ser Met Phe Ser Gln Ala Met
    605 610 615
    aag aaa tgg gtg caa gga aat act gat gag gcc cag gaa gag cta gct 2042
    Lys Lys Trp Val Gln Gly Asn Thr Asp Glu Ala Gln Glu Glu Leu Ala
    620 625 630 635
    tgg aag att gct aaa atg ata gtc agt gac gtt atg cag cag gct cag 2090
    Trp Lys Ile Ala Lys Met Ile Val Ser Asp Val Met Gln Gln Ala Gln
    640 645 650
    tat gat caa ccg tta gag aaa tct aca aag tta tga ctcaaaactt 2136
    Tyr Asp Gln Pro Leu Glu Lys Ser Thr Lys Leu *
    655 660
    gagataaagg aaatctgctt gtgaaaaata agagaacttt tttcccttgg ttggattctt 2196
    caacacagcc aatgaaaaca gcactatatt tctgatctgt cactgttgtt tccagggaga 2256
    gaatggggag acaatcctag gacttccacc ctaatgcagt tacctgtagg gcataattgg 2316
    atggcacatg atgtttcaca cagtgaggag tctttaaagg ttaccaa 2363
    <210> SEQ ID NO 34
    <211> LENGTH: 662
    <212> TYPE: PRT
    <213> ORGANISM: Homo Sapien
    <400> SEQUENCE: 34
    Met Arg Gly Ala Gly Pro Ser Pro Arg Gln Ser Pro Arg Thr Leu Arg
    1 5 10 15
    Pro Asp Pro Gly Pro Ala Met Ser Phe Phe Arg Arg Lys Val Lys Gly
    20 25 30
    Lys Glu Gln Glu Lys Thr Ser Asp Val Lys Ser Ile Lys Ala Ser Ile
    35 40 45
    Ser Val His Ser Pro Gln Lys Ser Thr Lys Asn His Ala Leu Leu Glu
    50 55 60
    Ala Ala Gly Pro Ser His Val Ala Ile Asn Ala Ile Ser Ala Asn Met
    65 70 75 80
    Asp Ser Phe Ser Ser Ser Arg Thr Ala Thr Leu Lys Lys Gln Pro Ser
    85 90 95
    His Met Glu Ala Ala His Phe Gly Asp Leu Gly Arg Ser Cys Leu Asp
    100 105 110
    Tyr Gln Thr Gln Glu Thr Lys Ser Ser Leu Ser Lys Thr Leu Glu Gln
    115 120 125
    Val Leu His Asp Thr Ile Val Leu Pro Tyr Phe Ile Gln Phe Met Glu
    130 135 140
    Leu Arg Arg Met Glu His Leu Val Lys Phe Trp Leu Glu Ala Glu Ser
    145 150 155 160
    Phe His Ser Thr Thr Trp Ser Arg Ile Arg Ala His Ser Leu Asn Thr
    165 170 175
    Met Lys Gln Ser Ser Leu Ala Glu Pro Val Ser Pro Ser Lys Lys His
    180 185 190
    Glu Thr Thr Ala Ser Phe Leu Thr Asp Ser Leu Asp Lys Arg Leu Glu
    195 200 205
    Asp Ser Gly Ser Ala Gln Leu Phe Met Thr His Ser Glu Gly Ile Asp
    210 215 220
    Leu Asn Asn Arg Thr Asn Ser Thr Gln Asn His Leu Leu Leu Ser Gln
    225 230 235 240
    Glu Cys Asp Ser Ala His Ser Leu Arg Leu Glu Met Ala Arg Ala Gly
    245 250 255
    Thr His Gln Val Ser Met Glu Thr Gln Glu Ser Ser Ser Thr Leu Thr
    260 265 270
    Val Ala Ser Arg Asn Ser Pro Ala Ser Pro Leu Lys Glu Leu Ser Gly
    275 280 285
    Lys Leu Met Lys Ser Ile Glu Gln Asp Ala Val Asn Thr Phe Thr Lys
    290 295 300
    Tyr Ile Ser Pro Asp Ala Ala Lys Pro Ile Pro Ile Thr Glu Ala Met
    305 310 315 320
    Arg Asn Asp Ile Ile Ala Arg Ile Cys Gly Glu Asp Gly Gln Val Asp
    325 330 335
    Pro Asn Cys Phe Val Leu Ala Gln Ser Ile Val Phe Ser Ala Met Glu
    340 345 350
    Gln Glu His Phe Ser Glu Phe Leu Arg Ser His His Phe Cys Lys Tyr
    355 360 365
    Gln Ile Glu Val Leu Thr Ser Gly Thr Val Tyr Leu Ala Asp Ile Leu
    370 375 380
    Phe Cys Glu Ser Ala Leu Phe Tyr Phe Ser Glu Tyr Met Glu Lys Glu
    385 390 395 400
    Asp Ala Val Asn Ile Leu Gln Phe Trp Leu Ala Ala Asp Asn Phe Gln
    405 410 415
    Ser Gln Leu Ala Ala Lys Lys Gly Gln Tyr Asp Gly Gln Glu Ala Gln
    420 425 430
    Asn Asp Ala Met Ile Leu Tyr Asp Lys Tyr Phe Ser Leu Gln Ala Thr
    435 440 445
    His Pro Leu Gly Phe Asp Asp Val Val Arg Leu Glu Ile Glu Ser Asn
    450 455 460
    Ile Cys Arg Glu Gly Gly Pro Leu Pro Asn Cys Phe Thr Thr Pro Leu
    465 470 475 480
    Arg Gln Ala Trp Thr Thr Met Glu Lys Val Phe Leu Pro Gly Phe Leu
    485 490 495
    Ser Ser Asn Leu Tyr Tyr Lys Tyr Leu Asn Asp Leu Ile His Ser Val
    500 505 510
    Arg Gly Asp Glu Phe Leu Gly Gly Asn Val Ser Pro Thr Ala Pro Gly
    515 520 525
    Ser Val Gly Pro Pro Asp Glu Ser His Pro Gly Ser Ser Asp Ser Ser
    530 535 540
    Ala Ser Gln Ser Ser Val Lys Lys Ala Ser Ile Lys Ile Leu Lys Asn
    545 550 555 560
    Phe Asp Glu Ala Ile Ile Val Asp Ala Ala Ser Leu Asp Pro Glu Ser
    565 570 575
    Leu Tyr Gln Arg Thr Tyr Ala Gly Lys Met Thr Phe Gly Arg Val Ser
    580 585 590
    Asp Leu Gly Gln Phe Ile Arg Glu Ser Glu Pro Glu Pro Asp Val Arg
    595 600 605
    Lys Ser Lys Gly Ser Met Phe Ser Gln Ala Met Lys Lys Trp Val Gln
    610 615 620
    Gly Asn Thr Asp Glu Ala Gln Glu Glu Leu Ala Trp Lys Ile Ala Lys
    625 630 635 640
    Met Ile Val Ser Asp Val Met Gln Gln Ala Gln Tyr Asp Gln Pro Leu
    645 650 655
    Glu Lys Ser Thr Lys Leu
    660
    <210> SEQ ID NO 35
    <211> LENGTH: 162025
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: GenBank AC005730
    <309> DATABASE ENTRY DATE: 1998-10-22
    <400> SEQUENCE: 35
    gaattcctat ttcaaaagaa acaaatgggc caagtatggt ggctcatacc tgtaatccca 60
    gcactttggg aggccgaggt gagtgggtca cttgaggtca ggagttccag gccagtctgg 120
    ccaacatggt gaaacactgt ctctactaaa aatacaaaaa ttagccgggc gtggtggcgg 180
    gcacctgtaa tcccagctac tcaggaggct gaggcaggag aattgcttga acctgggaga 240
    tggaggttgc agtgagccga gatcgcgcca ctgctctcca gcctgggtgg cagagtgaga 300
    ctctgtctca aaaagaaaca aagaaataaa tgaaacaatt ttgttcacat atatttcaca 360
    aatttgaaat gttaaaggta ttatggtcac tgatatcctg tttcattctt tatataatca 420
    ttaagtttga aatgtatact tgcactacta acacagtagt taatcttagt cctacaagtt 480
    actgctttta cacaatatat tttcgtaata tgtatgcact ggtgtttatg tacgtgttta 540
    tgtttatatc tgttaaaatt agcagtttcc atctttttct attttgtacc atcacatcag 600
    ttcagaagga ttgacagagc aaaatgattt gatgaagtat aaaagtcaca tggtgagtgg 660
    cataaataca actctgaaca attaggaggc tcactattga ctggaactaa actgcaagcc 720
    agaaagacac atatcctata tgtcaagaga tgtaccaccc aggcagttaa agaagggaag 780
    tacacataga aagcacaatg gtgaataatt aaaaaattgg aatttatcag acactggatt 840
    catttgctcc taaagtcaga gtcctctatt gtttttttgt ttttgtgggt ttctttttaa 900
    atttttttat tttttgtaga gtcggagtct cactgtgtta cccgggctgg tctagaactc 960
    ctggcctcaa acaaacctcc tgcctcagct tcccaaagca ttgggattac agacatgagc 1020
    cactgagccc agcccagacg ctttagcatt tatgaagctt ctgaaatagt tgtagaaacc 1080
    gcataagctt tccatgtcac tttcaaagtt tgatggtctc tttagtaaac caaccaagtt 1140
    attcctcaag ggcaaaataa catttctcag tgcaaaactg atgcacttca ttaccaaaag 1200
    gaaaagacca caactataga ggcgtcattg aaagctgcac tcttcagagg ccaaaaaaaa 1260
    aggtacaaac acatactaat ggaacattct ttagaagagc cccaaagtta atgataaaca 1320
    ttttcatcaa agagaaaaga gaacaaggtg ttagcaaatt cctctatcaa ataacactaa 1380
    acatcaagga acatcaatgg catgccatgt ggaagaggaa gtgctagctc atgtacaaac 1440
    cagtagataa tttcaacttg ctgccgaatg aaacctcttt gcaaggtatg aatcagcact 1500
    tctcatgttt gttttgcttt gttttgtttt gtttttagag acaggccctt gctctgtcac 1560
    acaggctgga gtgcagtggc acgatcagag ctcactgcaa cctgaaactc ctgggctcaa 1620
    gggatcctcc tgccttagcc tcccaagtag ctgggactac aggcccacca tgcccagcta 1680
    attttttaaa ttttctatag agatgggatc tcactagcac ctttcatgtt tgatgttcat 1740
    atacaacgac caaggtacaa tgtggaaaag ggtctcaggg atctaaagtg aaggaggacc 1800
    agaaagaaaa ggggttgcta catagagtag aagaagttgc acttcatgcc agtctacaac 1860
    actgctgttt tcctcagagc agagttgatg atctaaatca ggggtcccca acccccagtt 1920
    catagcctgt taggaaccgg gccacacagc aggaggtgag caataggcaa gcgagcatta 1980
    ccacctgggc ttcacctccc gtcagatcag tgatgtcatt agattctcat aggaccatga 2040
    accctattgt gaactgagca tgcaagggat gtaggttttc cgctctttat gagactctaa 2100
    tgccggaaga tctgtcactg tcttccatca ccctgagatg ggaacatcta gttgcaggaa 2160
    aacaacctca gggctcccat tgattctata ttacagtgag ttgtatcatt atttcattct 2220
    atattacaat gtaataataa tagaaataaa ggcacaatag gccaggcgtg gtggctcaca 2280
    cctgtaatcc cagcacttcg ggaggccaag gcaggcggat cacgaggtca ggagatcgag 2340
    accatcctgg ctaaaacggt gaaaccccgt ctactaaaaa ttcaaaaaaa aattagccgg 2400
    gtgtggtggt gggcacctgt agtcccagct actcgagagg ctgaggcagg agaatggtgt 2460
    gaacctggga ggcagagctt gaggtaagcc gagatcacgc cactgcactc cagcctgggc 2520
    gacagagcga tactctgtct caaaaaaaaa aaaaaaaaaa aaagaaataa agtgaacaat 2580
    aaatgtaatg tggctgaatc attccaaaac aatcccccca ccccagttca cggaaaaatt 2640
    ctcccacaaa accagtccct ggtgccaaaa aggttgggga ccgctaatct aaataatcta 2700
    atcttcattc aatgctaaaa aatgaataaa ctttttttta aatacacggt ctcactttgt 2760
    tgcccaggct ggagtacggt ggcatgatca cagctcactg tagcctcaat cacccaggcc 2820
    ccagcgatcc tcccacctaa acttcctgag tagctgggac tacaggcacg caccaccatg 2880
    cccagctaat ttttaaattt tttatagaga tgggggtctc accatgttgc ccagactggt 2940
    ctcaaaccct gggctcaagt gatcctccct caaactcctg gactcaagtg atcctccttc 3000
    cttggcctcc caaagtgctg ggattacaag catgagccac tgtacccagc tggataaaca 3060
    ttttaagtcg cactacagtc atggacaatc aggcttttca acatgcagta tggacagtga 3120
    gtcccagggt ctgcttttcc atactgaaat acatgtgata ctaaggagaa aggtgctcgc 3180
    aaggatattt aaaatgaaga atatttaaaa tgaggaaaaa actgtttctt catgactttg 3240
    ataaggctga taaagaccat ttctgtgatc tcaggtgatt cactcaagta gtatatttca 3300
    gtaatcatta tctggaacag cctgaatctt aaccaaaata ccatgatttt ttaatgctgt 3360
    tatgatacct tgatgatatg accaaactgc aatgtaggca gctaaatctc cacgagtttg 3420
    acttccccga gagttgacag ttttcttcac aaattaaaga aatatatttt ttgatacatg 3480
    attggcatat ttaaaaacta cactgaaatg ctgcaaaatg atataaagaa acattttcca 3540
    gaatcaaatg caatcaaaga gtggattagg aatctactca ccattatcaa ctaaatagaa 3600
    acacttggac tgggtgtggt ggctcacatc tgtaatctca gcactttggg aggccaaggc 3660
    aggtggattg cttgaggcca ggagctcaag accagcctga gcaacatagc aaaactctgt 3720
    ctctacaaaa aaaaaaaaaa attaaccagg catggtggca gatgcttgta atcccagcta 3780
    ctctggaagc tgaagtagga ggactgcttg agcccaggag atcaagactg cagtgagccg 3840
    tggtcatgct gcgccacagc ctgagtgaca gagagagacc ctgtctcaaa aacaaaaaca 3900
    aacaaaaaac acttaacctt cctgtttttt gctgttgttg ttgttgtttg tttgttttga 3960
    gatggagtct cactctgttg cccaggctgg agtgcagtgg cgtgatcttg gctcactgca 4020
    agctctgcct cccgggttca cgccattctc ctgcctcagc ctcccgagta gctgggacta 4080
    taggcgcccg ccaccacgcc cggctacttt tttgcatttt tagtagagat ggggtttcac 4140
    cgtgttagcc aggatggtct tgatctcctg acctcgtgat ccacctgcct cggcctccca 4200
    aagtgctggg attacaggca tgagccaccg cacccggcca acctttctgt tttttagttt 4260
    gatatgcttg ttaactcagc agctgaaaga atgctgaaag tggccttcag taaaaaaatt 4320
    tcactagaat ctctacatcc atatttaatc tgaatgcata tccagattga tcagttagag 4380
    caaaaacact catcatcatt cctgatgacc tctaattctg gtttcggctt tctatttcaa 4440
    tggaaacaga ataaggaaag aaatggaagg gctctggaaa tttgtcctgg gctatagata 4500
    ctatcaaaga tcaccaacaa taagatctct cctataaata taaaacaagt ataattaatt 4560
    ttttaattat ttttttctct tcagaggatt ttatttcaag ataaaacata acttctaccc 4620
    atactattga ttccaaaggt tagaaaaagt gtttttcctc atcttatcct tcaaagaggt 4680
    cacagcaatg caaacatcta taaaatgcct ctgcataatt gtcagaagct atagtccaga 4740
    aatcattgaa aatgcttttc cattttaagc ttaggtgagg tgtcttagga aacctctatg 4800
    acaacttact ctatttattg ggaggtaaac tcccagactc tcccagggtc tcctgtattg 4860
    atctcatttt ttaggcttcc taatcccttg aagcacaatc gaaaaagccc tggatctctt 4920
    ttctgcacat atcatcgcgg aattcattcg gcttccagca agctgacact ccatgataca 4980
    agcggcctcg cccttctccg gacgccagtc cttgctgcgg ttagctagga tgaggggttt 5040
    gctgggcttc agtgcaggct tctgcgggtt cccaagccgc accaggtggc ctcacaggct 5100
    ggatgtcacc attgcacact gagctcctgg caggctgtac caatttttta attatttaat 5160
    atttattttt aaaattatgg tgaatatttt ggtattctgc tctaaaatag gcccataaat 5220
    gcacagcaga tatctcttgg aacccacagc tttccactgg aagaactaag tatttttctt 5280
    ttaaagatgc tactaagtct ctgaaaagtc cagatcctct acctctttcc atcccaaact 5340
    aagacttgga atttatgaga gatctagcta acagaaatcc cagacacatc attggttctt 5400
    cccagagtgc agtcctccta aagaggctca gccctaagca ggcccctgca ccaggagggt 5460
    gggtctgaga cccacatagc acttcccaag gtgcatgctc cagagaggca ctgaaacagc 5520
    tgagcacaag cctgcaagcc tggagaactc tcacagtcag aacggagggg gcccagtggg 5580
    actaacataa agagaaaagg gaacacagag aaatggatgg caccaacaac cagcaaagcc 5640
    ttcatggcca atgaaagcat cagtgacggg gccagaaccc tcatccccaa agactcttca 5700
    ctgcctttag tgaaaaacaa tggctagaga gtgaagttat gatcatgtat agagaggtaa 5760
    agttacattt ttatattctg actctgctaa tgtgaaattc cctatctgct agactaaaag 5820
    tttcagacac cctgttcaaa tatcccatta gttgctagag acttaaaatg aacagaacgc 5880
    acattgtcag gatgactatt accaaaaaat caaaagacag caagtattgg tgaggatgta 5940
    gagaaactgg aacttttgtg cactgtttat gagaatgtaa aatggagcag ctgctgtgga 6000
    aaagagtatg caggttcctc aaagagtaaa accaagatgt ggaaacaact aaatgcccat 6060
    cagtggatga aggggtagac aatatgtggt atatacatac catggagtac tattcagcct 6120
    ctaaaaaaaa aaaaggaaat tctataacat gcaacagcat ggatgaatct tgaggacatt 6180
    ttgctaatga aataaggcag tcatagaaag acaaatactg cacgactcca cttatatgag 6240
    ataccaaaaa tagacaaatt catagaatca aagagtacaa tggaggttac ctggagctgc 6300
    agggcgggaa acgaggagtt actaatcaac gaacataacg ttgcagttaa gtaagatgaa 6360
    taagctctca agatcagctg tacaacactg tacctagagt caacaataat gtattgtaca 6420
    cttaaaaatt tgttaagggt agattaacaa atgtagtaga tccacaaatg tggttaagtg 6480
    ttcttaccac agtaaaataa aaaaagaata tcaagcccag gagttcgaga ctagcctggg 6540
    taacatggtg aaaccctgtc tctacagaaa atacaaaaat tagccagctg tggaggtgca 6600
    ctcctaggga ggctgaggtg ggaggcttgc ttgagcccag gaggtcaagg ctgcagtgag 6660
    ccatgattgc accactgtac tccagcccag atgacagagc aagacaccac cccccccaaa 6720
    aaaagaaaaa gaatatcaaa cattttaaaa gatcagatac gcaagaacaa caacaaaaaa 6780
    gagatgaaca gagcatcgac cctcatctag tgggattctt ggtctaactg aaaaacagac 6840
    attgagagac aaacaatgac agtgatgtga tcacagcaat tacacaggta tcccctgggg 6900
    actgcagaag aaaggaggaa tgcctaactt tcagaaaata gagaaagcgt caaacagttg 6960
    gtgaaagcct tccaaaacta gagagaactg cacacaccaa atcacagaaa gaagaaaagc 7020
    cgtgggagat tctgggaccc accggctatt tttgatggct gaacaccctg ctgcaggaga 7080
    gacaggagct ggaaagcatg gtgggatgaa acctcaaaca gctttgcctg cattgcttaa 7140
    gatgactggg cttgattaac tctagtcaat ggggacaatt caatcaaaga agaaagatgc 7200
    tcaaattcac attttagaat gattttttat ggcagtatgg ggaatagatt aaaagagagt 7260
    gaagctggag gcaagaaact tgttaagagg caactgaaac agtctagatg ataaataata 7320
    aactgacaga gtgactagaa aaatcagaac aggctgaatc aacagatacc tagatgaaaa 7380
    taacaggact tgatcaccag ttgtatcttg gagaggaagg agttgtttcc ttgctttccc 7440
    tacgactggg aatacggaag gtttgccgtg tgtattggtt atatactggt gtgtagccaa 7500
    tcactgacaa ccatttagca gcttaaaaca caaaggctta tctcccagtt tctgtgggcc 7560
    aggaatctaa gataggctta gctggctggt tctggctcag agtttctcaa gaggttgcaa 7620
    tcaagatgtc agctggggtt gcatcatctg aaggctcaac tggggccgga gggtccactt 7680
    ccaaggagtt cactcacctg cctgacaagg cagtgctggt tgttggcagg agatctcaat 7740
    tcattgccaa gtgagcctct ctatagcatt gctggaacat cctccccatc tggcagttgg 7800
    cttctctcag catgagtgat ctgagagaga gagcaaggag gaagccacag tgttcttcct 7860
    actcctactc ctaacactat ggacctactc ctaacactct cacttctgcc ttattccatt 7920
    agttagaaag ggaactaagc tccacctctt gaaataagaa gtgtcaaaga atttgtggat 7980
    atatttaaaa atcatcacac tgtggaagtg gatagggggt tcaattaatg ctgaacttga 8040
    aatgcctgag acattcaaat gtccaacagg caatgaacat acccatagat ggtcatgact 8100
    ttagcaagaa tagaggaaga tcacagaatt aaggaggaat tgaaaggtaa aagaagtgga 8160
    gtcagattcc ccctgaaaag tgagccatga aaggaacttt aactattgag ttagaggtca 8220
    gagtaggaaa tttcggtgga attctttttt aaagaaagga accatataag catgttttga 8280
    ggtagaggga gaataaatca gtagacaggg agaggtaaaa aacataaatg ataggggata 8340
    gttgacaaag gtcttggcag aatcccttac ccattgactt ggggccaaga gagggacact 8400
    tctttgtttg agggataagg aaaataagaa agaatgggtg ctatttagtg tggtcctgtc 8460
    tctagggcaa acgcataggt aacaaactgt gtgtgttagg aatatagatg tgacctcaca 8520
    ttgagattct cacctcaaat ccattttgtt gttacctgta ccttcctacc ttctcttttt 8580
    gctacatgca gactgctgtt ttgtcttcct ggcctgttcc aggtttcagc attctggcat 8640
    atctgctacc ctgttcccaa acctctctag agtccatgct ccttccttgg atagtgtttg 8700
    attgggccac gtatctaaga agtgatgcct tcagttaggc ctgagaacct cctctatgga 8760
    aatctccatc agtgaccctg acagacttgg tatcttggag atgtcactgc tcccagcctg 8820
    tggtctagga gaatctcagc ctgggcctct agtagtatgg ataaggcgtt aaggtatctt 8880
    tgaaccagag tctgtcatat tcctcaatgt gggacagata aaacagtggt agtgctggtg 8940
    tttctgagct agaactctgg tttttggtct agattctttg atgtatgacc tttcagaggt 9000
    attaaaattt gttctaatac aatgttcaat acaaatgtag ttccttttct gttaggacct 9060
    caacaaaaca tgaccaactg tagatgaaca ttaaactatg acaattcatg gaaatgaata 9120
    cagtaatacc tgcggttccc ccattttagc agtcactatg gtgacatttg gcacaaatgg 9180
    ctatttaagg gtgcttttgt taaaacctac catcttacta ggcacatgat attgaaacta 9240
    atgaaataat ggagaaactt cttaaaaact tttaatgaat aaagtgatga agtgataata 9300
    ttttagctgc tatttataaa gtgactatta caggtcaaac attcttctag ggtttttttg 9360
    ttgaagttgt cacatttaat ccttaataac ccactatgag tcaggtattc ttctctcccc 9420
    tttggacagt tggggaaatg ggggtcagag aggttaggta atttgctcag ggccacacaa 9480
    cctgcatgta gaaaatctga gatttgtaca ggaacgtatc aaactctgaa gtccatgctt 9540
    ctattttccc atgctgcctt tctaataaaa ggtaactaat gctactggat gctgccccca 9600
    aagtgagtca ctttcacccc accctacttg attttctcca taaaactaat cacatcctga 9660
    caacttattt attgctgatc tcccccacta gattataaac tcaataaaag caagatcctt 9720
    gtctgctgaa tatcagtacc taaaacgctg tctagcacag agcaagtaat taatatttgt 9780
    tgaatgaaca aataaaggaa aaaaattcaa aggaagaaaa agccctaaaa cagatgttta 9840
    cctaaacata cattttaaaa gaaagcatat aacaaattca ggacagaatt taaatttgat 9900
    tttttaaaga aataaccaag tgctagctgg gcacagtggc tcacacctgt aatcctagca 9960
    ctctgggagg ccgaggcagg cagatcactt gaggtcaaga gttcaagacc agcctggcca 10020
    acatggtgaa acctgtctct actaaaaata cagaaattat ccaggcatgg tggcaggtcc 10080
    ctgtaacccc agctactcag gaggctgagt caggagaatt gcttgaaccc aggaggcaga 10140
    ggttgcagtg ggccaagatt gcaccactgc actccagcct gagtaacaaa gcaagactct 10200
    gtctgaagga gaaggaaaga aagaaggaaa gaaggaaaga aggaaagaag gaaagaagga 10260
    aagaaagaaa gaaagaaaga aagaaagaaa gaaagaaaga aagaaagaaa gaaagaaaga 10320
    aagaaagaaa aagaaagaaa gaaagaaaga accaagtgct tatttgggac ctactatgct 10380
    atgtttttcc atgcacgcta ttttcagtaa agcagttagc aaacttgcaa gatcataaca 10440
    acaaatatat gcttctataa ctctaaaatt gtgctttaag aagttcctct ttaccagctc 10500
    atgtatgcat tagttttcta agagttacta gtaacttttt ccctggagaa tatccacagc 10560
    cagtttattt aaccaaagga ggatgcttac taacatgaag ttatcaaatg tgagcctaag 10620
    ttgggccagt tcatgttaat atactccaga acaaaaacca tcctactgtc ctctgacaat 10680
    tttacctgaa aattcatttt ccacattacc aaggagccag ggtaggagaa tatagaaaga 10740
    ccacccaaga atccttactt ctttcagcaa aatcaattca aagtaggtaa ctaaacacat 10800
    gccctaacaa tgaatagcag attgtgctca gaagaatgat ctacaacatc ttactgtgaa 10860
    ggaactactg aaatattcca ataagacttc tctccaaaat gattttattg aatttgcatt 10920
    ttaaaaaata ttttaagcct aaattttaaa aggtttgata ttggtacatg aatagacaaa 10980
    cagacatgga ctagaccaag aattaggttc aaacatatac aggaatttaa tatacgataa 11040
    atctagtatt ccaaaggaac caacaaatgg tgttcagaca gcaggatagg catcaggaaa 11100
    aacacagttg ggcaccctac cttactccta acaccaggag taactgaagg agcaccaaat 11160
    atttatttat tttaattata gttttaagtt ctagggtacg tgtgcacaac atgcaggttt 11220
    attacatagg tatacatgtg ccatgttggt gaggagcacc aaatatttaa aagaaaaaaa 11280
    ttggccaggg gcggtggctc acacctgtaa tcccagcact ttgggaggcc aaggtgggca 11340
    gatcacctga ggtcgggagt tcgagaccag cctgagcaac atggagaaac cccatctcta 11400
    ctaaaaatac aaaattagcc aggcatggtg gcacatgcct gtaatcccag ctacttggga 11460
    ggctgaggca ggagaatagc tttaatctgg gaggcacagg ttgcggtgag ctgagatatt 11520
    gcactccagc ctgggcaaca agagcaaaac ttcaactcaa aaaaattaat aaataaataa 11580
    aaataaagaa agaaaagaaa aaaatgaaaa tagtataatt agcagaagaa aacaccgtag 11640
    aatcctcgga ctcttaggat ggggaatgcc tataatataa aaaccctgaa gttataaaag 11700
    agaaaatcac ctacatacaa accaaatctt tctacatgcc taaaacatag cacaaacaca 11760
    gctaaataat catagctgaa tgaactggga aaacaaaact tgactcatat ccagacagag 11820
    ttaattttcc tacacataaa gagtacctat ataaacccaa caaaaaaacc accactaacc 11880
    caaaataaaa atgtgacagg taatgaacag gtagttcaca gagaatacaa atggctcttc 11940
    ggcacataag atgctcagac tgacttttac ttatttattt tttgagagac agggtctcac 12000
    gatgttgccc aggttaggct caaactcctg ggctcaaatg atagtaccag gactacaggt 12060
    gtgccccacc gcacctggct cctcaaccac ctgtattaac aggaaatgca aaataaaact 12120
    ttcaaatcta ttttacctat tagaatggca aaaatttgaa aaacttcaaa catcatcatg 12180
    ttggtgagaa tgtgaggaga ctggcactct cattttttgc tgatagcata tatatactga 12240
    tggcttctat ggaaagcaat ctggcagcgt ctatcaaatg tacaagtgca tatatccttt 12300
    gacaaagcaa ttccactcta ggaatgtgtt ctatatggtt gtgcttcctg gggctgggaa 12360
    ctgggagcta agggacaggg gcagaagata atcttctttt ccctccttcc ccgttaaaca 12420
    tgttgaattt tatatactgt aatatattat ttttcacaaa agataatttt taagcgatat 12480
    gtctgggaat tttttttttt cttttctgag acagggtctc actctgtcat ccaggctgga 12540
    atgccatggt atgatctcag ctgactgcag cctcgacctc ctgggttcaa gcaatcctcc 12600
    cacctcagcc tcctgagtag ctgggactac aggcacgtgc catcatgcta atttttgtat 12660
    atacagggtc tcactatgtt gcccaggcta atgtcaaact cctaggctca agcaatccac 12720
    ccacctcagg ctccaaagtg ctgggattac aggcgtgagc caccgcgcct ggccctggga 12780
    attcttacaa aagaaaaaat atctactctc cccttctatt aaagtcaaaa cagagaagga 12840
    aattcaacct ataatgaaag tagagaaggg cctcaaccct gagcaacaaa cacaaaggct 12900
    atttctgaga caggaatttg ctgaacaaaa tcgagggaag atgacaagaa tcaagactca 12960
    cttctcggct gggcgcagtg gctcacacct gtaatcccag cactttggga ggccgaggcg 13020
    gacagatcac gaggtcagga gattgagacc atactggcta acacagtgaa acccagtctc 13080
    tactaaaaat acaaaaaatt agccgggcgt ggtggcaggt gcctgtagtc ccagctactt 13140
    gggaagctga ggcaggagaa tggcgtgaac ccaggaagcg gagcttgcag tgagccgaga 13200
    tcacgccact gcactccagc ctgggtgaca gagcaagact ctgtctcaaa aaaaaaaaaa 13260
    aagactcatt tctctagatc ttgagccgta ttcaaattta tctcagctta gtgagaggtt 13320
    aaagcaagga atatccttcc ctgtgggccc tgctccttac tgaaggaagg taacggatga 13380
    gtcaaggaca ccaatggaga aaagcactaa caccattatc tgatgaacat tacgtgaaga 13440
    agggtaagaa gtgaagtgga attgctgaag aagtcagtga aagcggacat tcatttgggg 13500
    aaatggaata taggaaatcc ataaaagtga ttaaaaagat gttagaggct gaggcggggg 13560
    gaccacaggg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc catctctact 13620
    aaaaatacaa aaaattagcc aggcgtggtg gcaggcacct gtagtcccaa ctactcggga 13680
    gactgaggca ggagaatggc atgaacctgg gagacggagc ttgcagtgag ccgagatcac 13740
    gccactgcac tccagcctgg gtgacagagt gagactccat ctcaaaaaaa aaagttagat 13800
    acgagagata aagatccaac agacacacaa ctgctaattc tgaacagaac aaaacaaatg 13860
    gcacaggaaa agaaaattta agatataaca ccggaaaact ttcctgaaat tgagtaactg 13920
    aatctatagc ttgaaagggt ttagcatatg ccaagaaaaa tcagtagagt ccaaccagca 13980
    caagacacat ctagcaaggc tggtgattct accaacacag agaaagaagt gggtgaccca 14040
    taatgcggaa aaaggcagac catctgcagt cttctccaga acactggagt ctgaagacaa 14100
    aagaatgctg cctactgagc cagaagggag agaaagtgac ccaacacatc tttaccaagt 14160
    tagaatgtca cgcattattt aaaggctgca aaagccatga aagacatgaa agaacacaag 14220
    catttacaac atgaaagaac acaagcattc tcatactcaa gaatccttaa gaaaaatgta 14280
    gtcctaatcc agcccactga aagttaaatg tacttaatgt gctcattaat gggaacttca 14340
    tagcttcaaa tcagtctggt cccatctacc aacatctctc gcccggcttt cctgcaatag 14400
    tcagcacctt tccctcctcc cagtcttgtc ccctggagtc tgctctcagc atagcagagt 14460
    gaccacatca acacccaagt cagagccctc cagtgcgcac tggtctacaa agcccttccc 14520
    accccccacc ccacgtgccc tccggatcct tgtgacgtgt ctcctgcata ccctagcagc 14580
    cctggcctcc tcactgcccc tcctgtacat caggaaggcg actccttgag tcttggctct 14640
    ggccgcctcc tccacctgca gtgagttaac tcccttacct actctaggtc attgctcaaa 14700
    tgtcagcatc tcaatggggc cctccctgac taccctattt aaattctaca tactcccctt 14760
    gaccccatgg acctcactca ccctattcca cttttattct tacaatttag cacttgttct 14820
    cttctaacgt attctaagac ttactcattt attacattgt ttgccacccc ctctagtaca 14880
    taaactccag aggggcaggg atttctgtct atttattcat ttctttatcc ctaggacata 14940
    gaacagggca tagttcagag tattcaatgt tatcaatgaa tgaactagca gtagtaccag 15000
    ttccagttag gcacagaatt aaatctaaat agaattaaat ctcatggtct gggttaacta 15060
    tggatagaaa attagatata attttaagaa gcctagaaag aaaaaattaa taatgtaaaa 15120
    ataatattaa tttgataata ataacaaaaa ctctgccagg cactgtggct caaatctgca 15180
    atcccagcta ctcaggaggc tgaggtggaa ggatcacttg agaccagagt tcaagactca 15240
    gcctaggcaa cacggcaaga aactgtctct aaaaaaatta aaacttaaat ttttaaaaaa 15300
    gaattctcaa agcgtcacaa aaactggaga ttaaggtaca ggaagtgtga agtaatatta 15360
    ctatgctaat ggtttttttt ttttttagaa aggtataacc aaaagatttc tttctcaagt 15420
    cgataaactg agaaagataa gcatatcttc caattaacag agggggagga aaagccagat 15480
    acaacaaaat aagatataaa ttagtttcca gttgaaaaca agagtaggag ttattttgca 15540
    tcacctcacc tgtgacctcc cccagcccaa aaaacactac tgataaacag ggtagaaaag 15600
    catcatctca gataaagcag gaaaaactgc cacagtctca aaccacaaac tataagcaca 15660
    cacctggcca accctgccaa gtctgggctc agtaggagga acgtgctgag agctaggatg 15720
    taccaactta gacattctgt gggatacaga tgtccctgga agggtcacac catctcaaag 15780
    gcacctgtaa tgcccactga ttacagccac catatgtgag agagaaactc agggcactta 15840
    gagagtataa caagaacctt atgtcatctg agatgaggaa tcctcagccc tgcaaattaa 15900
    ccaactcttt agaacaactg gcaaaacata aatatccaca acttttgttt cagtaattcc 15960
    actcttagat atcaatccaa agtacatgag acagcagata cacacacaaa atggtattta 16020
    ctgcagcatt gtttataata gcaaaaaaca agaaataatc catatgtctc aataggatac 16080
    tgggtacatg agggtatgta cccatcattc aaccatcaaa aagagtgata tggatgtcca 16140
    cagatggaca taaaaagctg tgtgttacgt gaaaacaaac tcaagcagca gcaggatggg 16200
    cttatgatag tcagtatgag ctaatttctg gaaaaaaaaa tctagtgtgt gcacagaaaa 16260
    catctgaaag aacagaaaca aaactatcag cagaatattg agatgtttta ctaagttgta 16320
    tatctatact gcttgtaatt tttaccccaa gcaagaatta ctttttggaa aaagaaaatt 16380
    caggaaataa agcatttctt taaacttcat gtttaaacaa atggtgatgg aataaaagag 16440
    ttcttattca tcataaacac acacagcaca catgcacgca tgtgcgtgag cacacccttt 16500
    acttgataaa taccatgttg aatattttag tctttccttt taggttctat cccttcactc 16560
    aaaatgcggt tataaataaa tgtacttttc atgtgccttc tgcctaaacc cactttaata 16620
    taactttaca gtcccattat cattatagtc tcaaagctag actcagcctg aaactaccct 16680
    ttcatttgga acccttatta aaatgccaca tacagctcct tcaaataaaa acaaacccta 16740
    ggacctgaca ctaggcttcc tttgttgcta ctcataatgg ccaagttctg tgcttataat 16800
    acatcttctt tcattttatt gctacatatc caagggtttt atatgttttt cttattatat 16860
    cttaattcaa aacaccatca cgctcttttc cagatgaaaa taaggaaaag aaattgagca 16920
    actgactgac ttaaaggtca taaaactata tagtagcaga gtcagcaaaa gaagaaacac 16980
    acatctccca agtagaggct gaaaaccagt accattcacc tccagggtga gctatataca 17040
    gattacaaag tcaccttctc taaatgttca aactgaatcc catacccata ctttaccact 17100
    acctcgtaag aacagcctca gatcttgtta tagccttttt tttagcatgc tgaagccaat 17160
    aaaatgcttc ccattcagca agagaaacaa gttctgaaac actgaataat ctgcccaggg 17220
    cctatgaaca tttccactgt gagaaatgtt ctccactgtg tggagaagat ccttactctt 17280
    ctccacacag gcagaacatt agaaaaattc ttggattcta tgatgcacag cttaggagtc 17340
    tgtttagcac aatttaagtc caaatagtta ttaaatcctc ctctgttcca gaaacagtgc 17400
    taaatactgt gaatataaaa attgaaaaga tactctcctg gctcccaaga aagtcagcca 17460
    gatagaggag acacaggcac acaaatcact gtcacatgaa gctctacctc cctaacttca 17520
    aacgagggcc taagtcacca agaatacagt agcagttgtg actacgagta actactataa 17580
    ttcaatactt tatcttccct tagaaaactc ttctcccttg gaaatttatt tgcatttcta 17640
    aataccattc cttactaaaa ggaagcaggg ctccttgggg aaatagctga ttctaggtgt 17700
    ggactatgaa atgaaaatgg tgagtctggg acatcccatg ttgcccagaa atcaaggaac 17760
    tgcccaaaga ttaacagagt catgttaaat ggacctaaga gtgaaccaga aggagctcac 17820
    tttgccccgc gtggaacaat ttcaagaaaa acatgacagt aatgaattat aaaacatgaa 17880
    ttaaaataca tattggtact aaaaagagaa caaaaggatg tggctttgga taaagctctt 17940
    cttcatggaa gaataccagc taataaatgt aaaggaaatg agagaattag aaaaattatc 18000
    attttgtaaa ccttaatata ttcacctaga catgctaaaa ccactgagta aaaggctgct 18060
    tgggaagagg atgctcacat gatctcagag tttcacacca cagataattt attagataca 18120
    ggaaggaaga tgtgatcaag cttcctgtga cccccagcca ggccccacaa cactatgtgc 18180
    ctccttgtga tgtgggagct acacagcatc gcccacacag cttctcgcca aaactgtttg 18240
    aagctaatca caagggaaga actggacagc ttctgaccat gagacgctcc accagacaac 18300
    ttgcttggcc tctccaaaga aacttgcttg gcctctccaa agaaaactca gtttcattta 18360
    aaaacaaaac taattattta aaaacaaacg aaaagcaagt tgtggacttg agctccaggg 18420
    acagagcaga catacttttc cctgttcttc ccagtaagtg gtaataaaaa ccctcaacac 18480
    tagatataaa acaaatataa gaaggttctg gaaggggaag aggaggcaga ctatccaggt 18540
    gccttgaggc ccacagaaca acccagtgat gggttcactg ggtcttcttt ttgcttcatt 18600
    atctcagact tggagctgaa gcagcaggca acttcaaaac accaaggggc acagattgaa 18660
    aagccccaag aaaagcctgc cctctctagc caaaggacca ggaaggagac agtctaatga 18720
    gatggaacac atttagacag taactgccca tttaccagca ataactgagc agggagccta 18780
    gacttccagt cttgtgagga cgtaccaagg tacccaacac ccccaccaag gctgagtaag 18840
    gactgcgact tttatccctg catggcagta gtaaggagcc catccctcac ccgccagcag 18900
    tgtcagggga acctggactt ccactcccac ccaggagtga tgaggccctc cctgctgggg 18960
    tcatgtcaga ggaggcctag tggagattca gtgacttaac cttttcccag agataatgag 19020
    gccacctttc ctccctcttc ccccatggtg acagtgaaag cactgtggca agcagtaggc 19080
    actcctaccc ctcctagcca gggaggtatc agggaggcca agtagggaac cagaataccc 19140
    acaaccaccc agcagcaaca ggggtccccc accccattgg gtgtcaatgg aagcagagcg 19200
    gaaagcctgg atatttaccc ccatctagaa gtaacaagct gatgtccccc ttcttctact 19260
    acaatggtgt tcaaaacagg tttaaataag gtctagagtc tgataacgta atacccaaat 19320
    cgttgaagtt ttcattgagg atcatttata ccaagagtca ggaagatccc aaactgaaag 19380
    agagaaaaga caattgacag acactagcac taagagagca cagatattag aactacctga 19440
    aaggatgtta aagcacatat cataagcctc aacaggctgg gcgcggtggc tcacgcctgt 19500
    aaccccagca ctttgggagg ccgaggcagg tggatcacaa gatcaggaga tcgagaccat 19560
    cctggctaac acggtgaaac cccgtctcta ctaaaaatac aaaaaaaaat agcaaggcat 19620
    ggtggtgggc acctgtagtc ccagctactc gggagcctga ggcaggagaa tggcatgaac 19680
    ctgggaagag gagcagtgag ccgagatcgc accaccgcac tccagcctgg gcaacagagc 19740
    aagacttcgt cccaaaaaaa aaaaaaaaaa aaaaaaaagc ctcaacaaac aactacaaac 19800
    gtgcttgaaa caaatgaaaa aaaaatcttg gcaaagaaat aaaagatata tattttggcc 19860
    aggtgcagtg gctcacagcc tgtaatccct gcactttggg aggctgaggc aggcggatca 19920
    cctgaggtca ggagtttgag accagcctga ccaacatgga gaaaccccgt ctctactaaa 19980
    aatacaaaat tagccagtca tggtggcaca tgcctgtaat cctagctact caggaggccg 20040
    aggcaggaga atcgcttgaa ctcaggaggt ggaggttgcg gtgagccgag atcccgccat 20100
    tgcacattgc actccagcct gggcaacaag agcaaaactc catctcaaaa aaatagatac 20160
    atattttaat ggaaatttta gaattgaaaa atacagtaac caaattgaat ggaaagacaa 20220
    catagaatgg agggggcaga caaaataatc agtgaacttc aacagaaaat aatagaaatt 20280
    acccaatatg aagaacagaa agaaaataga ctggccaaaa aataaagaag aaaaaagagg 20340
    agcagcagga ggaatgatgg aaaaagagaa aggaaggaag gaagggaagg agggagggaa 20400
    ggagtgaggg agaaagtctc aaagacctct gagactaaaa taaaagatct aacacttgtc 20460
    atcagggtcc aggaaagaga caaagatggc acagctggaa acgtattcaa aaaataatag 20520
    ctgaaaactt cccaaatttg gcaagagaca taaacctata gattcgaaat gctgaacccc 20580
    aaataaaaag cccaataaaa tccacaccaa aatacatcat agtcaaactt ctgaaaagac 20640
    gaaaagagaa aacgtcttga aagcagtgag tgaaacaaca cttcatgtat aagggaaaaa 20700
    caattcaagt aacagatttc ttacagaaat taaggaagcc agaaggaaat gacacaatgg 20760
    ttttcaagtg ctgaaagaaa agaagtgtca acacaaaatt ctagattcag taaaaatatc 20820
    cttcaagaat caatgggaaa tcaagacagt ctcagataaa gcaaaataag agaatatgtt 20880
    gccagcagat ctcccctaaa ggaatggcaa aaggaagatc atgcaacaga ccaaaaaatg 20940
    atgaaagaag gaatccagaa acatcaagaa gaaagaaata acatagtaag caaaaataca 21000
    tgtaattaca ataaaatttc tatctcctct taagacttct aaattatatt gatggttgaa 21060
    gcaaaaatta taaccctgtc tgaagtgctt ctactaaatg tatgcagaga attataaatg 21120
    gggaaagtat aggtttctat acctcattga agtggtaaaa tgacaacact gtgaaaagtt 21180
    acatacacac acacacgtaa gtatatataa atatatgtgt gtatatgtgt gtgtatatat 21240
    atatatacat ataatgtaat acagcaacca ctaacaacac tatacaaaga gataataacc 21300
    aaaaacaatt tagataaatt gaaatggaat tctaaaaaat attcaaatac tctacaggaa 21360
    gacaagacaa aaagagaaaa aaagaggagg acaaactaaa ttttttaaaa acataaataa 21420
    aatggtagac ttaagcccta acttatcaat aattacataa atgtaaatga tctaattata 21480
    tcaattaaaa gacagagata gcagagttaa tttaaaaaca tagctataag aaacctgctt 21540
    tgggctgagt gcagtgactc acacttgtaa tcccagcact tcgggaggcc aaggcgggtg 21600
    gatcacctga ggtcaggagt tccagaccag cctggacaac atggtaatac cccatctcta 21660
    ctaaaaatac aaaaaaatta gccaggcatg gtggcacacg cctgtagtcc caactactca 21720
    ggaggctgcg acacaagaac tgcttgaacc cgggcagcag aggtagcagt gggccaagat 21780
    tgcgccactc cagcctgaac gacagagtga gactccacct cagttgaaaa acaaaaaaga 21840
    aacctgcttt aaatatacca acatatgttg gttgaaatta aaagaataaa atatatcatg 21900
    aaaacattaa tcaaaagaaa ggagtggcta tattaataac ataaaataga cttcagagaa 21960
    aagaaaattt caagagacag gaataaaagg atcaagaaaa gatcctgaaa gaaaagcagg 22020
    caaatcaatc attctgcttg gagattcaac accctctctt aacaactgat agaacaacta 22080
    gacaaaaaaa tcagcatgga gttgagaaga acttaacacc actgaacaac aggatctaat 22140
    agacatttac ggaacactct acccaacaat agcaaaataa acattctttt caagtattca 22200
    ctgaacatat ccttagaccc taccctgggc cataaaacaa agctcactag tgattgccga 22260
    aggcttggat ggacagtgga agagctgcat ggggagggag aaggtgacag ttaaagagtg 22320
    taggatttct ttttgggata atgaaaatgt tccaaaattg attgtggtga tgttggcgca 22380
    actctacaaa tataaaaaag gccattgaat tgtacgtttt aagtgggtga aacatatggt 22440
    atgtggatta tatctaacgc tttttaaaaa cttaacacat ttcaaagaat agaagtcata 22500
    cagagtgtgc tctactggaa tcaaactaga aagaggtaac tggaggataa cgagaaaagc 22560
    ctccaaatac ttgaaaactg gacagcacat ttctaaaatc atccgtgggt caaagatatt 22620
    catttctgat attcattttt attgtttaat gtatttttaa aaatttctta agggaaataa 22680
    actgactaaa aatgaatatg gctgggtgcg gtggctcacg cctgtgatcc cagcactttg 22740
    ggaggccgag gctggtggat cacaagatca ggagttcgag accagcctgg ccaagatggt 22800
    gaaaccccgt ctcaactaaa aaactacaaa aagtagccaa gcgcagtggc gggagcctgt 22860
    ggtcccagct acttgggagg ctgaggtagg agaatcgctt gaacacaggc agcagaggtt 22920
    gcagtgagcc aagattgtgc cactgcacgc cagcctgggc gacagagact gcctcaaaaa 22980
    aaaaaaaaaa aaaaagaata tcaaaatttg tgggacatag ttaaagcaat gctgagaggg 23040
    aaatttataa cactaaatgt ttacattaga aaagagaaaa agtttcaaat caatagtctc 23100
    cactcccatc tcaagaacac agaagatgaa gagcaaaata aacccaaagc aagcaaaaga 23160
    aagaaaatat aaaaataaat cagtaaaatt gaaaacagaa acacaataaa gaaaatcagt 23220
    gaaacaaagt actgattctt cgaaagatta ataaaattga caaacctcta gcaaggctaa 23280
    caaacaaaaa agaaagaaga cacggattac cagttattag aatgaaagca taattagaaa 23340
    caactctaca cattataaat ttgacaatgt agatgaaatg gactaattac tgaaaaaaca 23400
    caaattacca caactcaccc aatatgaaat agataattgg gatagcctga taactactga 23460
    gaaaattgaa tttgtaattt taacactctt aaaacagaaa cattaaactt aatattttat 23520
    aaatattaga taaggtaatt atacccttcc ttaacaaata aaaacgacaa attattttgc 23580
    agctaaagag atgtatgtac tgtgaaaaat atcttcagaa aaatagaact ttgtttgaag 23640
    aataaggatt taaaaaatgt ttttaactct caagaagcaa atatctgggc ccagatggtt 23700
    tcactgaaga attctaccaa atgtttaatg aagaattacc accaactcta catagcatct 23760
    ttgagaaaac tgaagagaag ggaacatctc ccagttcatt ttatgaagtg ggtgttactc 23820
    tgatactaga actgtataag gacagctact cttgacacac tgcctatggg tagctctgct 23880
    ctgcaggaac agtcagaaaa aaaaaaaaaa gaagcactgg acaagggcag tataaaaaaa 23940
    gaaaactggg ccaggtgcag tggctcacac ctgtaatctc agcactttgg gaggctgacg 24000
    ctggtggatc acctgaggtc aggagtttga gactagcctg gccaacatgg taaaaccctg 24060
    tctctactaa aatacaaaaa ttagccaggc agggtggtgg ggaaaataaa aaggaaaaaa 24120
    aaacaaaaat aaactgcaga ccaatatcct tcatgagtat agacacaaaa ctccttaaac 24180
    tccttaacaa aatattagca agtagaagca atatataaaa ataattatac accatgatca 24240
    agtgggactt attccagaaa cgcaagtctg gttcaacatt tgaaaacaag gtaacccact 24300
    atatgaacgt actaaagagg aaaactacat aatcacatca atcaatgcag aaaaaagcat 24360
    ttgccaaaat ccaatatcca ttcatgatac tctaataaga aaaataagaa taaaggggaa 24420
    attccttgac ttgataaagc ttacaaaaga ctacaaaagc ttacagctaa cctatactta 24480
    atggtgaaaa actaaatgct ttcccctacg atcaggaaca aagcaaggat gttcactctc 24540
    attgctctta tttaacatag ccctgaagtt ctaacttgtg caaaacgata agaaagggaa 24600
    atgaaagacc tgcagattgg caaagaagaa ataaaactgt tcctgtttgc agatgacatg 24660
    attgtctcat agaaaatgta aagcaactag gggtaggggg gcagtggaga cacgctggtc 24720
    aaaggatacc aaatttcagt taggaggagt aagttcaaga tacctattgc acaacatggt 24780
    aactatactt aatatattgt attcttgaaa atactaaaag agtgggtgtt aagcgttctc 24840
    accacaaaaa tgataactat gtgaagtaat gcatacgtta attagcacaa cgtatattac 24900
    tccaaaacat catgttgtac atgataaata cacacaattt tatctgtcag tttaaaaaca 24960
    catgattttg gccaggcaca gtggctcata cctgtaatcc cagcatttta ggaggctgag 25020
    gcgagcagaa aacttgaggt cgggagtttg agaccagaat ggtcaacata gtgaaatccc 25080
    gtctccacta ataatacaaa aattagcagg atgtggtggc gtgcacctgt agacccagct 25140
    acttgggagg ctgaggcacg agaattgctt gaacaaggga ggcagaggtt gcagtgagct 25200
    gggtgccact gcattccagc ctggtgacag agtgagactc catctcaaaa aaaataaaat 25260
    aaagcatgac ttttcttaaa tgcaaagcag ccaagcgcag tggctcatgc ctgtaatccc 25320
    accactttgg gaggccgagg caggcagatc acaaggtcag gagtttgaga ccagcctgac 25380
    caacatggtg aaaccccatc tctactaaaa aatatataaa ttagccaggc atgtgtagtc 25440
    tcagctactc aggaggctga ggcaggagaa tcacttgaac ccggaggcag aggttgcagt 25500
    gttgagccac cgcactccag cctgggtgag agaacgagac tccgtctcaa aaaaaaaaag 25560
    caaaataacc taattttaaa aacactaaaa ctactaagtg aattcagtaa gtctttagga 25620
    ttcaggatat atgatgaaca tacaaaaatc aattgagctg gacaaaggag gattgtttta 25680
    ggtcagtagt ttgaggctgt aatgcacaat gattgtgcct gtgaatagct gctgtgctcc 25740
    agcctgagca gcataatgag accacatctc tatttaaaaa aaaaaaaatt gtatctctat 25800
    gtactagcaa taagcacatg ggtactaaaa ttaaaaacat aataaatact gtttttaatt 25860
    gcctgaaaaa aatgaaatac ttacatataa atctaacaaa atgtgcagga cttgtgtgct 25920
    gaaaactaca aaacgctgat aaaagaaatc aaagaagact taaatagcgt gaaatatacc 25980
    atgcttatag gttggaaaac ttaatatagt aaagatgcca attttatcca aattattaca 26040
    caggataaca ttattactac caaaatccca gaaaaatttt acatagatat agacaagatc 26100
    atacaaaaat gtatacggaa atatgcaaag gaactagagt agctaaaaca aatttgaaaa 26160
    agaaaaataa agtgggaaga atcagtctat ccagtttcaa gacttacata gctacagtaa 26220
    tcaagactgt gatattgaca gagggacagc tatagatcaa tgcaaccaaa tagagaacta 26280
    agaaagaagc acacacaaat atgcccaaat gatttctgac aaaggtgtta aaacacttca 26340
    acgggggaag atatgtctct cattaaaggg tgtagagtca ttgcacatct ataggcaaaa 26400
    agatgaacct gaacctcaca ccctacagaa aaattaactc aaaatgactc aaggactaaa 26460
    cataagatat acatctataa aacatttaga aaaaggccac gcacggtggc tcacgctcgt 26520
    aatcccagca ctttgggagg ccaaggcagg tggatcacct aaggtcagga gtttgagacc 26580
    agccggatca acatggagaa gccccatctc tactaaaaat acaaaattag ctggacgtgg 26640
    tggcacatgc ctgtaatccc agctacttgg gaggctgagg catgagaatc gcttgaaccc 26700
    ggggggcaga ggttgcggtg agccaagatc acaccattgc actccagcct gggcaacaag 26760
    agcaaaactc caactcaaaa aaaaaaaaaa aaaggaaaaa tagaaaatct ttgggatgta 26820
    aggcgaggta aagaattctt acacttgatg ccaaactaag atctataagg ccagtcgtgg 26880
    tggctcatgc ctgtaattcc agcactttgg tcaactagat gaaaggtata tgggaattca 26940
    ctgtattatt ctttcaactt ttctgtaggt ttgacatttt tttagtaaaa aattggggga 27000
    aagacctgac gcagtggctc acacctgtaa tcccagcact ttgggaggcc ggggcaggtg 27060
    gatcacacgg tcaggagttc gagaccagcc tggccaacat ggtgaaaccc cgtctctacc 27120
    aaaaatataa aaaattagcc gggtgtcatg gtgcatgcct gtaatcccag ctactgagga 27180
    ggctgaggca ggagaatcac ttgaacctgg gaggtggaag ttgcagtgag ccgagattgt 27240
    gccactgcac tccagccttg ggtgacagag cgagactccg tctcaaaaga aaaaaaaaaa 27300
    aaagaatatc aaacgcttac tttagaaact atttaaagga gccagaattt aattgtatta 27360
    gtatttagag caatttttat gctccatggc attgttaaat agagcaacca gctaacaatt 27420
    agtggagttc aacagctgtt aaatttgcta actgtttagg aagagagccc tatcaatatc 27480
    actgtcattt gaggctgaca ataagcacac ccaaagctgt acctccttga ggagcaacat 27540
    aaggggttta accctgttag ggtgttaatg gtttggatat ggtttgtttg gccccaccga 27600
    gtctcatgtt gaaatttgtt ccccagtact ggaggtgggg ccttattgga aggtgtctga 27660
    gtcatggggg tggcatatcc ctcctgaatg gtttggtgcc attcttgcag gaatgagtga 27720
    gttcttactc ttagttccca caacaactgg ttattaaaaa cagcctggca ctttccccca 27780
    tctctcgctt cctctctcac catgtgatct cactggttcc ccttcccttt atgcaatgag 27840
    tggaagcagc ctgaagccct cgccagaagc agatagtgat gccatgcttc ttgtacagcc 27900
    tacaaaacca tgagcccaat aaaccttttt tctttataaa ttatccagcc tcaggtattc 27960
    ctttatagca agacaaatga accaagacag ggggaaatca acttcattaa aataatctat 28020
    gcagtcacta aacaaataag aacaagaggc tccagaagtg ggaagccaat acccagagtt 28080
    cctacaatac agtatctgaa aagtccagtt tccaaccaaa aaatatatat atacaggccg 28140
    gacatggtag cttatgtctg taatcccagc actttgggat gctgaggcgg gcagatcacc 28200
    ctaggtcagg agttcgagac cagcctggcc aatatggcaa aaccccgtct ctactaaaaa 28260
    tacaaaaatt agccaggcat ggtggtggat gcctgtaatc ccagctactc gggaggctga 28320
    ggcagggaat cacttgaacc caggaggcag aggttgcagt gagccgagat cacgccactg 28380
    aactccagcc tgggcaacaa agtgagactc cacctcaaaa aaaaaaaaaa tatacatata 28440
    tatatgtgtg tgtgtgtgtg tgcgcgcgtg tgtgtatata cacatacaca tatatacata 28500
    tatacagaca cacatatata tatgaagcat gaaaagaaac aaggaagtat gaaccatact 28560
    ttctgtggtt atgataggat ggggtatcac gggggaagta gacaagggaa actgcaagtg 28620
    agagcaaaca gttatcagat ttaacagaaa aagactttgg agtaaccatt ataaatatgt 28680
    ccacagaatt aaagaaaagc gtgattaaaa aaggaaagga aagtatcata acaatattac 28740
    tccaaataga gaatatcaat aaaggcatag aaattataaa atataataca atggaaattc 28800
    cggagttgaa aggtagaata actaaaattt aaaattcact agagaaggtt caacactata 28860
    tttgaactgg cagaagaaaa atttagtgag acaaatatac ttcaatagac attattcaaa 28920
    tgaaaaataa aaagaaaaaa gaatgaagaa aaataaacag aatctcagca aaatgtggca 28980
    caccattaat cacattaaca tatgcatact gagagtaccg gaagcagatg agaaagagga 29040
    agaaaaaata ttcaaatgat ggccagtaac ttcctagatt tttgttttaa agcaataacc 29100
    tatacaatca agaaactcaa tgaattccaa gtaggataaa tacaaaaaga accacaaaca 29160
    gatacaccat ggtaaaaatg ctgtaagtca aaaacagaga aaatattgaa agcagctaga 29220
    ggaaaactta taagagaacc tcacttacaa aagaacatca cttataaaag aaccacaata 29280
    atagaaacag ttgacctctc atcagaaaca atgaatgata acatatttga agtgctcaaa 29340
    gaaaaaaaat aaagattcct atatacgaca aagctgtctt tcaaaaatat acatccaaaa 29400
    ggattgaaac cagggtcttg aagagttatt tgtacatcca tgttcatagc agcattattc 29460
    acaatagcca aaaggtagaa gcaacccaag ggtccatcga caaataaata aaatgtggta 29520
    tatgtataca caatggaatt tattcagtat taaaaaggaa tgaaattctg acacatgcta 29580
    caacatggct aaaccttgag aacactatgc taagtgaaat aagccagcca caaaaggaca 29640
    aataccatat tacttcactt gtatgaaata cctagggtag tcaaattcag agatagaaag 29700
    taaaacagtg gttgccaagg gctgagggag ggagtaacgt ggagttattg ttgaatgggt 29760
    acagaatttc agttttgcaa gataaaaaga gttctggaga cagatggtgg tgagggtggt 29820
    acaacaatac aaatatactt tatactactg aacagtatac ttaaaaatga ttaacatggt 29880
    gaaaccccgt ctctactaaa aatacaaaaa aattagctgg gtgtggtggc gggcacctgt 29940
    aatcccagct acttgggagg ctgaggcagc agaattgctt gaaaccagaa ggcggaggtt 30000
    gcagtgagct gagattgcgc caccgcactc tagcctgggc aataagagca aaactccgtc 30060
    tcaaaaaata aaaaataaaa aaaatttaaa aatgattaag caggaggcca ggcacggtgg 30120
    ctcacaccta taatgccagc actttgggag gccgaggcag gcgatcactt gagaccagga 30180
    gtttgagacc agcctggcca acatggcaaa accctgtctc tgctaaaaat acaaaaatta 30240
    gccaggcatg gtggcatata cttataatcc cagctactgg tgagactgag acacgagaat 30300
    tgcttgaacc caggaggcag agattgcagt gagtcgagat cgcgccactg aattccagcc 30360
    tgggcgacag agcaagattc tgtctcgaaa aaacaaaaac aaaaacaaaa agcaaaacca 30420
    aaaaataatt aagcaggaaa cgagattgct gctgaggagg agaaagatgt gcaggaccaa 30480
    ggctcatgag agcacaaaac ttttcaaaaa atgtttaatg attaaaatgg taaattttat 30540
    atgtatctta ccacaaaaaa aagggctggg gggcaggaaa tgaaggtgaa ataaagacat 30600
    cccagagaaa caaaagtaga gaatttgttg ccttagaaga aacaccacag gaagttcttc 30660
    aggctgaaaa caagtgaccc cagagggtaa tctgaattct cacagaaaat tgaagcatag 30720
    cagtaaaggt tattctgtaa ctatgacact aacaatgcat attttttcct ttcttctctg 30780
    aaatgattta aaaagcaatt gcataaaata ttatatataa agcctattgt tgaacctata 30840
    acatatatag aaatatactt gtaatatatt tgcaaataac tgcacaaaag agagttggaa 30900
    caaagctgtt actaggctaa agaaattact acagatagta aagtaatata acagggaact 30960
    taaaaataaa attttaaaaa atttaaaaat aataattaca acaataatat ggttgggttt 31020
    gtaatattaa tagacataat acaaaaatac cacaaaaagg gaagaagaca atagaactac 31080
    ataggaataa cattttggta tctaactaga attaaattat aaatatgaag tatattctgg 31140
    taagttaaga cacacatgtt aaaccctaga tactaaaaag taactcacat aaatacagta 31200
    aaaaaataaa taaaataatt aaaatgtttg tattagtttc ctcagggtac agtaacaaac 31260
    taccacaaat tgagtggctt aacacaactt aaatgtattt tctcccagtt ctggaggcta 31320
    aacacctgca atcaaggtga gtacagggcc atgctccctg tgaaggctct aggaaagaat 31380
    cctcccttgt ctcttccagc ttccagtggt tctcagtaac cctaagtgct ccttggcttg 31440
    tagctatatc attcctagca accagaaaga agaaaataat aaagattatg gcaaaaaata 31500
    atgaaatcaa aaggagaaaa atggaaaaaa ataaataaaa ccaaaagcta gttctttgaa 31560
    aagatcaacc aagttaacaa accttttaac tagactgaca aaaaggaggt aagactcaaa 31620
    ttactagaat cagaaataaa agaggggaca ttactaatga gggattagaa aagaatacta 31680
    cgaacaaatg tgtgccaaca aattagaaaa cttagatgaa atggacaggt tcctaggaca 31740
    acatcaacta ccaaaattta ctcaagaaga aagagacaat ttgaatgagc tataacaagg 31800
    gaagagactg aattgacaac caagaaacta tccacaaaga aaatcccagg cccagaagat 31860
    ttcactgtga aattctttca aacttataaa tataaattaa catcagttct tcacaaactc 31920
    ctccaaaaaa aagaacagat ctctatttac aggcgatacg atctttagaa aatcctaagg 31980
    gaactactaa gacactatga taactgataa acaagttcag caaggctgca ggatagaaaa 32040
    ccaatataca aaaatctatt atatttctat acacttgcag tgaacaaccc aaaaatgaga 32100
    ttaagaaaat aattcaattt acaataacat caaaaagaat aaaaacactc aaaaataaat 32160
    ttattcaagt aagtgcaaaa cttatactct agaagctaca aaacactgtt aaaagaaatt 32220
    aaaggtttac ataaatgaaa aactatccca tgttcatgga tcaaaagact tattactggc 32280
    aatgctctcc aaattgatct ataaattcaa caaaatcctt atcaaaatcc cagatgaggc 32340
    tgggggtggc ggttcatgcc tgtaatccca gcactttggg aggctgaggc acgcagatta 32400
    cctgaggtcg ggagctcgag atcagcctga ccaacatgga gaaaccctat ctcttctaaa 32460
    aatacaaaat tagtcaggcg tggtggcaca tgcctataat cccagctact cgggaagctg 32520
    aggcaggaga atcgcttgaa cccaggaggc agaggttgca gtgagccaag atcgtgccat 32580
    tgcactccag cctgggcaac aagagcaaaa ttccatctca aaaaaaaaaa aaaaaaaatc 32640
    ccagatgact tcactgttga aattgaaaag attattctaa aattcacatg gaattgcaag 32700
    accttgagaa tagccaaaac aaacttgaaa aacacgaaca aaatatagga tgactcactt 32760
    gccaattgca aatgttacga cacagcaaca gtaatcaaga ctgtgtggta ctggcaaaag 32820
    acacatacat acatacatat caatggaata taattgagag tacagaaaca agcctaaaca 32880
    tctatggtaa gtgcttttct atttttttct tttttttttt cttttttgta gagatagaat 32940
    ctcaccatgt tgcccaggct ggtcttcaac ttctgggctc aagcaatcct cccactgtgg 33000
    cctcccaaag tgctgggata actggcatga gccaccacat ccagcccaga tgattttcaa 33060
    aaaagtcaac aagaccattc ttttcaacaa ataggtctgg gatgatcaga tagtcacatg 33120
    aaaaaaaaaa tgaagttgga ccctccatca cactaaagtg ctgcgattat aggcatcagc 33180
    caccacatcc agcccaaatg attttcaaaa aggtcaacaa gaccattctt ttcaacaaat 33240
    aggtctggga taatcagata gtcacatgaa aaaaaaaatg aagttggacc ctccatcaca 33300
    ccatatgcaa aaattaattc aaaaatgaat tgatgactta aacgtaagag ttacgactgt 33360
    aaaactctta gaaggaaaca tacgggtaaa tcttaaagac gttaggtttg acaaagaatt 33420
    cttagacatg acaccaaaag catgaccaac taaggtaaaa tagggtaaat tgtacctacc 33480
    aaaatgaaaa acctttgtgc tggaaaggac accatcaaga aatggaaagc caaaatagcc 33540
    aaggcaatat taagcaaaaa gaacaaagct ggaggcatca tactacctga cttcaaagca 33600
    acagtaacca aaacagcatg gtactagtag aaaaacagac acatagacca atggaacaga 33660
    ataaagaacc caaaaataaa tccacatatt tatagtcaac tgatttttga caatgacacc 33720
    ccttcaataa atgatactag gaaaactgga tatcgatatg cagaagaata aaactagacc 33780
    cctatctctc accatataga aaaatcaact cagactgaat taaagacttg aatgtaagac 33840
    ccaaaactat aaaactactg gtagaaaaca taaggaaaaa cgcttcagga cattggtcca 33900
    ggcaaagatc ttatggctaa aacctcaaaa acacaggcaa caaaaacaaa aatggaaaaa 33960
    tagcacttta ttaaactaaa aagctcctgc acagcaaagg aaacaacaga atgaaaagac 34020
    aacctgtaga atgggagaaa atatttgcaa actatccatc catcaaggga ctagtatcca 34080
    gaacacacaa gtgactaaaa caactcaaca gcaaaaaagc aaataatctg gtttttatat 34140
    gggcaaaaga tctgaataaa cattctcaaa ggaagacata caaatgtcac tatcattctg 34200
    ccagtaccac actgtcttga ttacttgtta gtgtataaat ttttaaattg ggaagtgtga 34260
    gtcatcctac actttgttct tgtttttcaa gtttgttttg gctattctgg gagccttgca 34320
    agtataaaat agccaacaag tatgaaaaaa tgctcaccat cactaatcat cagagaaata 34380
    aaaatcaaga ccactatgag atatcctctc actccagtta gaatggctac tatcaaaaag 34440
    acaaaatata atggatgctg gcaaagattt ggagaaaggg gaactcctat acactgtggg 34500
    tagggatgca aattggtaat ggccattatg gaaaataata ctgaggtttt tcaaaaaact 34560
    gaaaatagaa ctaccatatg atccagcaac cctactactg ggtatttatc caaaggaaag 34620
    aagtcagtat actgaagaaa tatatgcact ctcatgttaa ttgcaacact gttcacaaca 34680
    gccaagacag ggaataaatc taaatgtgca tcaacagatg aatggataaa gaaaatgtgg 34740
    catatacact caatagaata ctattcagcc attaaagaag aatgaaatcc tgtcatccca 34800
    gcaacatgga tgaacctgga ggacattata tttaatgaaa taagtaaagc acaaaaagat 34860
    aaacagtaca tgttctcact cagacatggg tgctaaaaag aaaatggggt cacagaatta 34920
    gaaggggagg cttgggaaaa gttaatggat aaaaatttac agctatgtaa gaagaataag 34980
    ttttagtgtt ctatagaact gtagggcgag tatagttacc aataacttat tgtacatgtt 35040
    caaaaagcta gaagagattt tggatgttcc cagcacaaag gaatgataaa tgtttgtgat 35100
    gatggatatc ctaattaccc tgattcaatc attacacatt gcatacatgt atcaaattat 35160
    cactctgtac ctcataaata tgtataatta ttacgtcaac aaaaaaagga aaaaaaagaa 35220
    aattaagaca acccacataa tggaagaaat aaaatatctg caaattatat atatctgata 35280
    aatatttaat atttataata tataaagaac tcctacaact caagaacaac aacaaaacaa 35340
    cccaattcaa aaatgggtaa aagccttgaa tatacactta tctaaagact atatacaatt 35400
    ggccaataaa gacacgaaaa gatgctcaac atcactagtc atcagggaaa tataaatcaa 35460
    aaccacaatg tagaatgtag acaccacttc atatgcacta ggatggctag aataaaaagg 35520
    taataacaaa tgttggtaag gatgtgaaaa aatcagaaac ctcattcgct gctgttggga 35580
    atgtaaagtg atgcagccac tttggaaaac agtctggcag ctcctcaaat tattaaatac 35640
    agagttaccg tatgacccag gaatattcct cctgggtcta taaccaaaaa aatgaaaaca 35700
    tatatccaca taaaaacttg tacatgggca tttatagcaa cattattcat aacagcaaag 35760
    gtggtaagaa cccatatgcc catcatctga tgaacaggta aataacatgc ggtattatcc 35820
    atacactaga atattatctg cccatacaag gagtgacatc cagctacatg ctacaaggat 35880
    gaatctcgga aaccttatgc taagtgaaag aagccagtca caaatgacca cagattatga 35940
    ttccatgcat cggaaatgac cagaataggg aaatctatag agacagaaag tagattagtg 36000
    gttgggtggg gctgggagga caggtagtac actactttcc cagaactact ggaacaaagt 36060
    accacaaact ggggagctta aacatagaaa ttgatttcct cacagttctg gagactagga 36120
    ctctgagatc aaggtgtcag cagagctggt tctttctgag ggccctgagg caaggctctg 36180
    tcccaggcct ctctccttgg ctggcaggtg gccatcttct ccctgcgtct tcacatcatc 36240
    ttttctctgt gtgtgcccat gtccaaattt tgattggctc attctgggtc atggccaatt 36300
    gctatgcaca aagtgaagtc tacttccaaa agaagggaag agggaacact gactaggcta 36360
    aacttatagt cattttaatg tccgcttttc ctatgagatt gtgaacacac agaagtaggg 36420
    tttttatcta cattgtgcaa agtttaataa gaaaaataga attcaagaga agcagttcaa 36480
    tagcaggaat ttaatatggg aactaattac aaggtttagg gcaggactaa aaagccagtt 36540
    gggatggtga gccaacccag agattagcaa cagtgggacc ccatctacct accacccatg 36600
    aagctggaag gataaaggag gggctattat cagagtccac aagccagtgt cagagtcctt 36660
    ggctggagct gggaccaccc tagagacact gtgcaaagca gaaaacaagg gggaaaaacc 36720
    ctgacttctc ccttcctccc acctttcaat ctcccactag tgcttcctac tagccatact 36780
    tggccagaga cagtgacaag gaacactgca aaatgaagtt tgtaggaatc atctccctct 36840
    gagacagaga aatatggaag ggtagaaaat gaatcagagg ataaagagaa aaaaccctga 36900
    gtactatctt atttatcttt gtatctccag tgcctaatct gtctctcaaa aaaggaaagc 36960
    aattgagaga aactgaaaac tccaattgaa atgaaagaat ggagaattac tggactagaa 37020
    gagaagagaa aaatttattc cgcatagagt aaacaagaat ggattcacaa aggacgtgat 37080
    gaatgaaaag ctataatcag caaagatttg ccagagaaat taaaaagtgg taaactcagc 37140
    cacgctgtac aacctgaagg cacaatgcat gaaaacgttt caagaaatga caagatttga 37200
    agtcaaattc taagtgcttt tccagaatct ctcaagacga ttatatagct accccatttt 37260
    attaaataaa atggaaactt actaaacttt ccccttgtat taaactaaca tatgtcctaa 37320
    tagcaaacga ttctggaatt cctagagtaa aatatatttc gtcaaagtgt attgctcttt 37380
    taatattctg ctgacctcct tttgctattt aggatatttg tatacacatc acacgtaaat 37440
    ttggtctata gtttacatct acgggcttat actgttcttt ttttcatttt tttaaaattt 37500
    ccaaccccca gtatccatat actgctctct atcagggtta ttttaacttt gtaaaatcag 37560
    ctgagatgct ttccatgttt ttttttttta ttttctgcca catttgaata gcataggagt 37620
    taccaccatc aaccttggat tatttaagca ttcacgattc cacgtgtgga ttttttattc 37680
    agagtctttc ttgtcattcc tgctatcagc acagaaccca atctcagctt tccagctata 37740
    ctctcacccc atggaatttg cagatgaagt tcaaaaggac ctttgcatta tcctgcctcg 37800
    ccctcttccc ccttcattta gacatcacct tcttctagaa cgtcttacct gacatgccct 37860
    gctcccaacc cctgctgccc aattgtgtgc tctcccgtgt cctggcctgc catcctcttt 37920
    agtaattgcc tgctccctca tctgtctccc cacccagaca ttaagctgaa tagactggat 37980
    ttgtgtcttg tccatcacta taatctcagc acctagtacc tagtaggtac ttaccatgta 38040
    ttcattagca aaatgttatg tataaccttg caccttaaaa acaagagaag gaagacaaaa 38100
    ttaagtctta agactatggt ttagaacatg gatcagaaac tacagtctgc agcccaaatc 38160
    cagaccaaat gaagagacca tgttcattta catacaacct atagcagctt tcacactaca 38220
    ggagcagagc taagtagttc caagggaaca cacggccctg caaagcctaa aatatttact 38280
    ctatagctct tcacagaaaa agttttcaga tccctcgttt agaactcttg ttcatatgca 38340
    atttcactaa accatagttt tttgggtttg tttggttttt tttggcaaaa aggaatgagc 38400
    cgatccagaa aaggttgaaa agaatgaatc attactgctg aaagaatgtg cacacagtcc 38460
    gtcagtattc tgctgccatg ctgacaccca tccaatagtg tcatgagatg cagcagctac 38520
    tactgtgttc tcaatgccga gtccacccac tccataacca tgtccaagca atcttgggaa 38580
    catcatcacc atgcttgttt atccttaagg tattgcctca catacagcag tggctggtca 38640
    taaagtcaaa tgacactagt ggccaggagg tcaagagaat gagtgaggac aggtgggtag 38700
    gcagcccagg ccctagcaac agcaggagct cacccctcag tcactctagc caggactgaa 38760
    atacttttca ccctttcaag agagactagg aatctggatt tttatgtgaa atatcttgat 38820
    tactaaatgt tgtcaacaga catgtcaaaa ggtaaaacta agtaagttca tggggcagat 38880
    tgactattca ggttatagaa ttaaggattc ttatccaaca cagataccaa ccaaaaagct 38940
    gacgtataac atattaggag aaactatgtg cactgtcgaa acatcaacaa ggggctaatg 39000
    tctaaaatag tctatattgg attccagttg aaacatgggg aaaggacatg aacaggcaac 39060
    ttatgtcaat ggaaactcaa aaagataaca agcatatata aaagcattct caaattcagt 39120
    agtaaacaga cagatgcaaa taaaaagagg gaaactgctg ccgggcacag tggctcacac 39180
    ctgtaatccc agcactttgg gaggccgagg cgggcggatc atgaagtcag gagatcgaga 39240
    ccatcctggc taacatggtg aaaccccgtc tctactgaaa acacaaaaaa ttagccaggc 39300
    gtagtggtgg gcaccagtag tcccagctac tcaggaggtt gaggcaggag aatggcatga 39360
    acccaggagg cggagattgc agtgagccga gaccatgcca ctgcactcca gcctgggcga 39420
    ctgagtgaaa ctccatctca aaaaatataa taataattat aattataata ataataaata 39480
    gtaaataaat aaaaagagag agactgctaa agtctagaaa gttgaatgat gccaagcgca 39540
    tgcaaagatc agggccttgg gatggccggg tgcagtggct cacgcctgta atcccaccac 39600
    tttgggaggc caaggcgggc ggatcatgag gtcaagagat caagaccatc ctggccgaca 39660
    cagtgaaacc cggtctctac taaaagtaca aaaaaatata tatatatata tatattatta 39720
    tattatatat atatatatca gagccttggg aatccttgtg tgctgctggg gaaggtagtg 39780
    gtgcagccac ccttgacagc aatctggcag tacttggtta tattaagtat aggcacacac 39840
    cacgaccagg cagtcctact cctgggtcta aatcccaaag aattctcaca caagtccata 39900
    aggagacatg tacgaggctc attcagcatt actgggagtg ggaatcaacc tgggtgtcca 39960
    tctacaggag acgagatgga caaaatgtgg tggatattaa gaccagaatc accaagtaac 40020
    agagatgggt ggtgagtgac aatcctaaga tacagaataa aggctagaac atgatgccat 40080
    tcatgtaaat taaaaataga tgcacacaaa gcagtatacg cgtgaccctt gaatagcaca 40140
    ggtttgaact gcctgtgtcc acttacatgt ggattttctt ccacttctgc tacccccaag 40200
    acagcaagac caacccctct tcttcctcct ccccctcagc ctactcaaca tgaagatgac 40260
    aaggatgaag acttttatga taatccaatt ccaaggaact aatgaaaagt atattttctc 40320
    ttccttatga ttttctttat ctctagctta cattattcta agaatatggt acataataca 40380
    catcacacgc aaaataaatg ttaattgact gtttatatta tgggtaaggc ttccactcaa 40440
    cagtaggctg tcagtagtta agttttggga gtcaaaagtt atacacagat tttcaactgt 40500
    gcaggcaatc agttcccctg accccctcat tgttcacggg tcaactgtat atacacaaaa 40560
    gtattatatg aacctcatta gaatagctgt ctatagggag aagagaatga gagtgggata 40620
    aaacggaatg aacaaataaa ccaacaaatg cattaacaag caaaacaaca gaggggcttg 40680
    catgggccag tgatgataaa gggctaagaa tgagaatata attaattcaa ttcctcacac 40740
    ctgaggtcta aaaccaagga aagggagggc caggcgtgga ggctcacgcc tgtaatccca 40800
    gcactttggg aggctgaggc gggcggatca caagattagg agtttgagat cagcctggcc 40860
    aacacagtga aagcccatct ctacaaaaaa tacaagaatt acccaggtgt ggtggcacat 40920
    gcctgtagtt agctactctg gaggctgagg caggagaatc acttgaaccc aggaggcgga 40980
    ggttgcaggg agccgagatc acaccattgc actccagcct gggtgacaga gtaagactct 41040
    gtctcaaaaa aataaaaaaa ataaaaaaac agagaaaggg aggaaactag atccaggctg 41100
    actagataca gcctttagag ttagaaaaga tgatttgaca atctaagccc acactcagat 41160
    tgaatgaaat tgaaaagcct ttcaaactaa aacatttaat tacaccatct gctgcagaca 41220
    gaactcagac aactcaaaca ggtaatgtca gcgtggtgtt ttatatcacc accctcaaca 41280
    cagaataaaa atcagctgca tgtgaagcag tgactagaat gaagaaaagg ctgcttctta 41340
    cttccttcta gtggttcttt ccgaaaacat taataggcac cagctctatg catgtcaccc 41400
    tgcagggaga catggggtat ataactatga cttactgttc attcctcaag gaattcccaa 41460
    tcttgtggaa gattatacac aatgaggcaa caaaaactat ccaataaaac cacggaaaag 41520
    aagccagtga caaagaagcc agtgatgaaa ggccctgtga gcagagctga tggccatttg 41580
    gggaagaaag accaacatgg atgggggtga tcagggtggc tccgtgggaa agctggaaga 41640
    gaagtggcag atctctgagc tggatgatgg gccactacca tctgtatatg gctaattaaa 41700
    gaccatgtgt ggatttttta ttcagctctt tcgtgtcatt cctgctatca gcacagaacc 41760
    caatctcaac tttccagcta tattgagcta aacttctcac ctcatggaat ttgcagataa 41820
    agttcaaaag gatccttgcc ttttcaaaat aattttgaat ggttgagtag tccctctgtg 41880
    ctctctcact gacaccctct caaggctgct gagcacgtgc catgctatgg ctttctccaa 41940
    catcaggaaa tgttctccac tcagtttcac cttaatacaa atgtgttctc tcttcagaga 42000
    aggcaaaaaa attcatgacc atctgactgg gagaagtcat ttctaggtaa agtgtccatc 42060
    tttttctgag gaacacagga ggaaaatctt acagaaaaga gttaacacag caggcctaag 42120
    actgcttttt aaaataaata aataaataaa taaataaata aataaataaa taaataaata 42180
    aataaatgaa tgatagggtc ttctgtattg gccaggctag tctcaaattc ctggcttcaa 42240
    gagatcctcc caccttggtc tcccacagtg ttgggattat agacatgagc cattgtgctt 42300
    ggcccaagac tgttattctt aaaaagtctc ataaaaagca tggttaatcc ttggctggca 42360
    cctgggaact tagatttcag aagggttccc accatccaac ctggaaagag ggactcactg 42420
    tgcctaaatt attgtgtggt ttatgctgaa ctcctgcttt tcttcaggta gcgtggaatg 42480
    tggtatgtgc tgggcaaagg gggcctgcat gaccagcccc caataaaaac cctgggtgtt 42540
    gggtctctag tgagtttccc tggtagacag catttcacat gcgttgtcac agctccttcc 42600
    tcggggagtt aagcacatac atcctgtgtg actgcactgg gagaggatgc ttggaagctt 42660
    gtgcctggct tcctttggac ttggccccat gcacctttcc ctttgctgat tgtgctttgt 42720
    atcctttcac tgtaataaat tacagccgtg agtacaccac atgctgagtc ttccaagtga 42780
    accaccagat ctgagcatgg tcctgggggc ccccaacaca gaaataaatt ataaaagacc 42840
    aaggactggg catggtggcc catgccggta atctcagcgc tttgggaggc cgaggcagga 42900
    ggaccagtta agcccaaaag ttcaaagtta cagtgaccta tgactgcgcc aatgcactct 42960
    aacctgggag acagagcaag accctgtccc caaaacaata aactaaacac atacttctgc 43020
    cttccaagtg tcttaaaatt caatggaatg gtagaaacat ttttaaaaca ctaaatcaaa 43080
    agaaacctgg aaaacaagag tgccgatggc caactaaaat gtctaggaaa tttctgaaaa 43140
    gtaaaaagta ctcagaacca gattacctga gcaaaccata gcccaataca agcttgggag 43200
    gaggctgtta tgcagaagga aatggtaaca ggtttccagg aacagacttg taacagcaga 43260
    tagaacagca gaggtagaac ctgacaaggt gattacctgg ggaactgcag tctgaatgac 43320
    caggactgtt ggacccttcc cctcacatgg aatacacacg ccactcagca gcacaccaca 43380
    gctcttcaac aatcacagga ggcacgctac gcctagtaag acaggaaaaa aggaattctc 43440
    aaacttcgaa gatgaacaca taaagaatca ccaagttttt attcagtatg atgaaacagg 43500
    gacactgaat caacagaaca caaacccaag caaagataat tactagagca catagaagaa 43560
    attattagat attcttggga agacctaagg ggacattata aagagcaagc agttggtatg 43620
    tgacgatctt tgtgatatac caagaaataa aaacacagga tgaagaccag atagagaata 43680
    atgctactat ttgtgcaaaa aaggagaaat ggagaatctg attcatattt gcttgtattt 43740
    gcatgaagaa actttggaag gtacataagt aactaacaac aatggttacc tacttgtaag 43800
    gcgagagaag taagaggaca ggaatggtgg gaacaccttt tgtgtccgga attggtgggt 43860
    tcttggtctg acttggagaa tgaagccgtg gaccctcgcg gtgagcgtaa cagttcttaa 43920
    aggcggtgtg tctggagttt gttccttctg atgtttggat gtgttcggag tttcttcctt 43980
    ctggtgggtt cgtagtctcg ctgactcagg agtgaagctg cagaccttcg cggcgagtgt 44040
    tacagctctt aagggggcgc atctagagtt gttcgttcct cctggtgagt tcgtggtctc 44100
    gctagcttca ggagtgaagc tgcagacctt cgaggtgtgt gttgcagctc atatagacag 44160
    tgcagaccca aagagtgagc agtaataaga acgcattcca aacatcaaaa ggacaaacct 44220
    tcagcagcgc ggaatgcgac cgcagcacgt taccactctt ggctcgggca gcctgctttt 44280
    attctcttat ctggccacac ccatatcctg ctgattggtc cattttacag agagccgact 44340
    gctccatttt acagagaacc gattggtcca tttttcagag agctgattgg tccattttga 44400
    cagagtgctg attggtgcgt ttacaatccc tgagctagac acagggtgct gactggtgta 44460
    tttacaatcc cttagctaga cataaaggtt ctcaagtccc caccagactc aggagcccag 44520
    ctggcttcac ccagtggatc cggcatcagt gccacaggtg gagctgcctg ccagtcccgc 44580
    gccctgcgcc cgcactcctc agccctctgg tggtcgatgg gactgggcgc cgtggagcag 44640
    ggggtggtgc tgtcagggag gctcgggccg cacaggagcc caggaggtgg gggtggctca 44700
    ggcatggcgg gccgcaggtc atgagcgctg ccccgcaggg aggcagctaa ggcccagcga 44760
    gaaatcgggc acagcagctg ctggcccagg tgctaagccc ctcactgcct ggggccgttg 44820
    gggccggctg gccggccgct cccagtgcgg ggcccgccaa gcccacgccc accgggaact 44880
    cacgctggcc cgcaagcacc gcgtacagcc ccggttcccg cccgcgcctc tccctccaca 44940
    cctccctgca aagctgaggg agctggctcc agccttggcc agcccagaaa ggggctccca 45000
    cagtgcagcg gtgggctgaa gggctcctca agcgcggcca gagtgggcac taaggctgag 45060
    gaggcaccga gagcgagcga ggactgccag cacgctgtca cctctcactt tcatttatgc 45120
    ctttttaata cagtctggtt ttgaacactg attatcttac ctattttttt tttttttttt 45180
    tgagatggag tcgctctctg tcgcccagac tggagtgcag tggtgccatc ctggctcact 45240
    gcaagctccg cctcccgggt tcacaccatt ctcctgcctc aacctcctga gtagctggga 45300
    ctacaggcaa tcgccaccac gcccagctaa ttttttattt tatttttttt ttagtagaag 45360
    cggagtttca ccatgttagc cagatggtct caatctcctg acctcgtgat ccatccgcct 45420
    cggcctccca aagtgctggg attacagacg tgagccactg cgccctgcct atcttaccta 45480
    tttcaaaagt taaactttaa gaagtagaaa cccgtggcca ggcgtggtgg ctcacgcctg 45540
    taaccccagc actttgggag gccgaggcgg gcggatcacg aggtcaggag atcgagatca 45600
    tcctggttaa cacagtgaaa ccccgtcgct actaaaaata caaaaaatta gccgggcgtg 45660
    gtggtgggca ccggcagtcc tcgctactgg ggaggctgag gcaggagaat ggcgtgaacc 45720
    tgggaggcag agcttgcagt gagccgagat agtgccattg ccttccagcc tgggcgacag 45780
    agcgagactc cacctcaaaa aaaaaaaaaa aaaatagaga cccggaaagt taaaaatatg 45840
    ataatcaata tttaaaaaca ctcaagagat gggctaaaga gttgacggaa caaatctaaa 45900
    tattagattg gtgacctgca aaaccagccc aaggaacatc ccagaatgca gcccataaag 45960
    ataaagagag catttccgct gggcacagtg gtatggcagg ggaattgcct gagtccaaga 46020
    gttgcaggtc acattgaacc acaccattgc actccaggcc tgggcaacac agcaatactc 46080
    tgtctcaaaa aaaaaaaaaa ttaaattaaa aaagacagaa tatttgagag aaaaaaatgc 46140
    ttatttcaag aaacatgaaa gataaatcaa gatattctaa ttcccaagta agaataattc 46200
    cagaagcaga aaatagaata gaggcaagga aacactcaaa acttctccag tgccatagaa 46260
    atgtgtatta atctttagaa tgaaacggac taccaaatgc tgagcaggaa gaacaaaaga 46320
    gatccactct taagccagtg tggtgcccaa gcgcagtggc tcatgcctgt aatcccagca 46380
    ctttgggagg ccgaggcagg tggatcacct gaggtcagga gtttgagatc agtcaggcca 46440
    acatggtgaa accctgtctg tactaaaaat acaaacatta gctgggtatg gtggtgcaca 46500
    tctgtaatcc caactacttg ggaggctaag gcaggagaat cacttgaaac caggaggtgg 46560
    aggttgtagt gagccgagat catgccacac tcccagcctg ggtgacagag caagattcca 46620
    tctcaaaaaa aaaatccact cctagacaaa taatagttaa attttagaac accaaggaga 46680
    aagaaaaaaa attgtaaagc ttcagagaaa ataaacatta actacaaaga aacgagagtc 46740
    agacgcgtgc acttcttcct agataccagc agataaagca atatctccaa aattcagaag 46800
    gttttaacgt agaatcctat acccagtcaa gaatattcac atggaaaagt gaaataaaaa 46860
    acattgttta aacatgcaag ggttcagaaa gtttaccatt cacagaatcc ctgaaaacaa 46920
    aaccaaataa tcacttaagg actcattaag aaaacaaatg aaataaaagc accaatgatg 46980
    agtaaataat cagaaaaatt tacagtttac ctaaataact gtttatgcat aatgtatgaa 47040
    aacccaaaaa tttaatatgg gacagaatta aaatcatgat aagattcttt tttgctttac 47100
    tcatggagag ttcacataaa cagattatct tttaatagca agagaaaaaa atgtttagat 47160
    atgtgtgaaa aactaagggt accaaaacag tgcaaattca tttatcatca ggaaaatcca 47220
    aattaaaacc acagtatcca ccagaataac taaaaggtaa aagacagaaa ttaccaagag 47280
    ttggcaagaa tgtggagcaa ccacatatac ttctggggta aataagttgg tgcaaccggt 47340
    actgaaaact gtttgctagt atctactaaa accgagcaca tgcacagact acaaccaagc 47400
    agttccactc ccagatacac actcaacaga aatgcacaca ctcactcaac aaaagacgtg 47460
    tactagagtg ttcatgtact tactattcat aatagtccaa aaatgcaaac aaccaactgc 47520
    caatcaaagt caaatgtata tctatattag ggatatatac aatggcatat acacagcaat 47580
    gagaatgaaa tgaaccagct cggcacagtg gttcatgcct gtaatctcag cactttgggc 47640
    gggtaaggca ggcagatcac ttgaggtcag aaatttgaga ctagcctggc caacacggtt 47700
    aaaacctgtc cccactaaaa acacaaaaat tagccgggca tagtggttgc aggcctgtaa 47760
    ttccagctac tcgggaggct gggttgggag aatcgtttga acccgaaagc cggaggtcgc 47820
    agtgagcgga gatcgtgcca ctgcactcca gcctggacga tagagcaaga ctccgtctca 47880
    aaaaaggaaa tcaaaaatat aaaataagat gacaggaata atccgcaaaa gatcagtaat 47940
    caaaataaat ataaatgggc taaagctacc tattaaaaga caaagatttc acacccataa 48000
    ggatagctac tatcaaaaaa agagagagaa taacagatgt tagcaaggat gtatggaaac 48060
    tgaaattctc acgcattgct ggtgagaata taaaatggtt cagcctctgc ggaaaacact 48120
    atgctgggtc atcaaaaaat taaaaataga agtactactt gatccaacaa ttctacttct 48180
    gggtatatac ccaaataact gaaagcaggg tcttgaagag atatttgtac acccatgatc 48240
    atggcagcat tattcataat agctatgatg tggaaccaac ataaatatcc tttgataaat 48300
    atatggataa gcaaaatgtg gtgtatacat tcaatggaat attaattagc aataaaaatg 48360
    aagaaaattc tgacacatgc tacaacatgg atgaaccttg agggcattac attaaatgaa 48420
    ataagccagt tataaaaaga caaatactat atgaggtact atattagata ctcatgcaag 48480
    gtacctaaaa taggcaaatt catagagaca aaaagcagaa tggtggttgc caggggctgc 48540
    ggtaatggat acagagcttc aattttgtaa gatgaaaaaa ttctggagat tggttgcata 48600
    acaatgtgca cacacttaac actggggaac tgtaaactta aaagtagtaa atggtaaaaa 48660
    taaaaataat aaataataaa ttttatgtta ttttaccaca atatttatta aaagacaaag 48720
    attaactaat taaacaaaat ccagccataa gctaatggta agagtaacaa ttaaagaaga 48780
    cacagaaaat tgaaaatcag tgactagaaa aagatattcc atataaatgc taacaaaaag 48840
    caagtacagc aatataaaga gaatgaacaa aaaaaaaatt aaataagatg gctcgtttat 48900
    tcccaaaagg tacaattcac caagaagata caagaattgt gaacctttaa gcacataaaa 48960
    cagcttcaaa aatacaacat ttaaagaaaa atatatatta aacatagaaa tagtacaaaa 49020
    acccctacaa gaatcataat gggagtcttc aatacaactc tccatatcaa caggtcaaac 49080
    agagaaaaaa aataagttaa ggatgcagaa aacctgaatt accatcaata aacttgagat 49140
    taatatagaa ctgtataccc aatatactaa gagttcaggg aacagtcgtg actgacagtg 49200
    gactgcaaat taatctgttc ttaatctttg tttttctttc agcactgtgg cagaatagag 49260
    atcctaaaaa ccttccagct acaaaacatc tttttaaaaa tataaaaaaa tacaaaaata 49320
    actctgaaat caatagaaga cacatggtga aaccaaaatt ctagaataca gggagaataa 49380
    aggcattttc agatattaca aaaacagaaa attgatcatt gctgaagtaa tttctaaaga 49440
    atgtacttga gggagaagaa aaatgttcca aagaaaagta tctgtgatac aagaaggaat 49500
    ggaaagtgaa gaaatggtaa acaggtagat aaagctaata aatgttgacc tagaaaataa 49560
    caaaaacaat agcaataatg tctcgttgga agggttgaag taaaaataca attaaggcca 49620
    aatgtgaggt aagtggaatg aaagaattag aagtccttgc cttgttcaca ggactgatta 49680
    aataaatgag ccaggttttc cattcaaaca gttaaaactt gaacaaaata aactcaaatt 49740
    aagtagaaag ataaaaaaca gaaattaatg tcatagaaaa ataaaaaatc aatagaatta 49800
    atcaataaat cctggttaat aaaagctggt tctttgaaag gattaataaa ataatcatta 49860
    agcaagtctg atcaaaaaaa aagagaaaag gtaccaaaaa aagtactgta tcagaaagag 49920
    aacatacaga tacatacaga tatgtaagag tctgttttct tacaccagaa tactatatac 49980
    aacattatgc tagcatatat taaatttcaa taatgttaat gattttctag gaaaacagaa 50040
    aatattaaat ttactttgaa gaaacagaaa aactgagaaa aataaatgat catgaaaaaa 50100
    atgaaaaggt aattaaatac tgatattaac tgcctaaaca acaccagcag cagcccaggc 50160
    agtctgcagt caagttctgc caaacttgag ggaacagata attcttctat tccagagcat 50220
    agaaaatgat ggaaagtttc ccaatttaat cagagaggac agcctgatcc ttgttatgaa 50280
    cacagataaa aatggggtaa actatatgcc aaactcagat accaaaaccc taaataagat 50340
    gctagcttat tgatgtgaac aatccaaaag tgcattttaa attagcccag ggttttagag 50400
    aaagaaaatc tagcaatgtg accaccactt atgttaacaa ttttaagacg aaaatctaca 50460
    tgatcatatc aatgcatgct acacaaaagc atttgggcaa aaaacccaac acccaccctt 50520
    gactttttaa actcttagta attaggcata aacagaaatg tacttaatgt gatagaatac 50580
    actcggtgaa gatacagagg gaatgctccc taaaaccaag cccaagacaa agattcctat 50640
    ttaacctcaa tagtcaacac tgcagcgaga gtaatctatg gaagacaagg aaaaaagtaa 50700
    aaacatgaga gacatctgtt gtttaacaga caataagatc acctacttgg aagaggcaaa 50760
    cgaatcaagc gaaaaactat taaaactgag acaggcttta gtatggaggc tcagcttcag 50820
    ctgtagtttg ggctaccaaa ttcaactcgc ttgcttggag agttaatcct gcaaagctaa 50880
    tttctgttga ggtattagga ttgacaagcc tgtgctcctc cctcctcccc catcttcaac 50940
    actgaaataa cacggtgttt ggaactggat aacagaatct tccaaaaaca aaaattgtcc 51000
    tgaagggctg acttgtgccc ttactcaaaa aacactttat ctgctgcctg cagctcctac 51060
    agttgctggt ggataagcct gccaaccagc tcggcgtaat tcttcctgca gagggcaagg 51120
    aagagcactt tcacaggaaa atttttttcc gaactgtatg ccgcttatta cataaactta 51180
    cgtgctggca aatggagctc cagcaaaata agatattcag agtcaaactt ccttaggaaa 51240
    aaaaaaaaaa aaaagcaagc acataacact aatttccttg catgggcact ggggaaggag 51300
    gtcgttactt ccgcacgccc gcaggtccgc accaccggga aacccacggg caccgcgcgc 51360
    tgcccccggg ccttccaggt gcactgcgcc gcggcgcccc agctgacccg ggatgcgcag 51420
    ccctagccct tcccctgtca ccccggccag gaaggggcgg gagcgcggcg gacgccgagg 51480
    gcgaagggct tctcggtcct ctgcaccacg cagcaccccc aaggcacaac agggagggtg 51540
    cgggaggctc ccgagaccca ggagccgggg ccgggcgtgc ccgcgcacct gtcccactgc 51600
    ggcgagggct ggggtcgcct ccagggccgc agctgtcggg agccacctgg ctctcagtcc 51660
    cgggtccctg cgacaaccct cgggcccgga ggggaggagg cggccacctg ccgctgccac 51720
    ctgcggcacc ggtcccaccg ctccgggccg ggcaggacag gccaggacgt ccctcctggg 51780
    ctggggacag gacacgcgac gaggggaccg gggcccccgc ggcgaagacg cagcacgcct 51840
    tcccagaaag gcagtcccgt gcccccacga cggactgccg gacccccgcg ctcgcccgcc 51900
    catcccttca gaccacgcgg ctgaggcgca aagagccggc cggcgggcgg gctggcggcg 51960
    cggctagtac tcaccggccc cgctggctca gcgccgccgc aacccccagc ggccacggct 52020
    ccgggcgctc actgatgctc aggagaggga cccgcgctcc gccggcgcct ccagccatcg 52080
    ccgccagggg gcgagcgcga gccgcgcggg gctcgctggg agatgtagta cccggaccgc 52140
    cgcctgcgcc gtcctccttc agccggcggc cgggggcccc ctctctccca gctctcagtg 52200
    tctcatctcc ctatctgctc atcctctggt cgcacataat cgatgtttgg gcgtcccaag 52260
    ccagatgtgg accccatttc cgcactctac actggaggtt ttctaagggt ggtgcccgga 52320
    ccagcagctt cagcctcatc tgggaacttg agaaaatgca gattctccgt cccacccagc 52380
    ctattcggtt tttcctgcac taaaaccatg aaggtggggc ccagcagtcc acattctcgc 52440
    aagcccgtca agtgattctg aggcgccctc cagtttgaga gctatgctca cggcctcacc 52500
    tccgccccgc aaggagcccg gtcttgcctg tggcgctagc cgcacacgga cacctcatcc 52560
    tgcggggccc gcccccccgc tgcaccctca ccgcccaacg cctcctccgg gatgcagcgg 52620
    aggcgcctgg aagtcggcaa ggtcaacatc cccctcagca tcttccctac cctcacggct 52680
    cctcctccag gggtgcctca tggccagggg ttagaaagag ccactgtgtt tcttgacatg 52740
    gaagtggcct aagaccttaa tgaaaactgc aggagtggaa tgacagaacc tttggtcata 52800
    cttgagggcg tgaagctcaa atgaggagga aggaaaggat ccagggagaa taaccaaccc 52860
    tggcaagttg tggcgcccag gtagaggggc gagcctaggc tagcggttct cgaccagggc 52920
    cggtgttgcc cctcctcgcc gccccgcgta catttgggga ggtctggaga catttttggt 52980
    tgtcatgatg cgggagttgc tactgttgcc taagtgggta gacacgaggg tgctcctcaa 53040
    catcctacct gaaggacagg actgccccac aaggaagaat gatccggccc caaataagaa 53100
    accctgggct ggtcagcaac aacccctttg ttctgagaag agaggaggaa agaataaaag 53160
    aagtggggtg aagttttggt ttggtagagg aaacttgaag acattttcac tggaaaggaa 53220
    gagaggaaga ggagggagat gtctgtaagg acgagcaaac cgggtgacag ctgatttcct 53280
    catattgaag taatgagtcc tagttataat aaattcctaa taaaaaccca gtttatccct 53340
    gcaataaact tgtctttttt ttttaaatat actgcttgat tctgtttgct aatattttat 53400
    ttacaggctt tgcattgata tgcaaaaatg agatgggcaa taattttctt tttgaatgtc 53460
    taatgttgtt tggtttcaga atcaatgtta tgctcacatc ataaaaaatt tggaaccgag 53520
    gcaggaggag tgcttgaggc cagaagttcg agaccagtct aggaaacaca gtgagacccc 53580
    cccatctcta caaaaaaaaa aaaagaaaaa aaaatgggca tgtttgcttt ttccttttac 53640
    tctgaacaat ttaaggagca ttaaaattat ctattctttg aggtttgatc atttcccagt 53700
    taaaaatgtt cctcccagcc tgatgctttc tttggggagg gtaaatcttt taaggctaga 53760
    aaagtttctt ctgtggcaat tttattattt acattttaaa aattattcta gagttaattt 53820
    tgataaagca tgtatttctt aaaacaaatt atcctttttt tccagatgtt caagtgtatt 53880
    tgcataaagt tgaggaaagt agtcttttgt gaatctttta acttctccca aatatcttat 53940
    tttgtgtatt tttgcttctt tattttgtta acttttaaaa gtgtattttt ttttcaaaga 54000
    atcagctctt aggtttatgt ttttggttat actggagctt ttttcttctt ctttttaaaa 54060
    tattttttct cctttatttt ttagacgtat tttgatctaa cgtaatcgga agaaggtaaa 54120
    ttagaatctt ttgttactat tgtgttttta tttctcctta tttctctgaa gtcctgcttt 54180
    ataaatagta ccatgttatt tgtgcataaa tattcatttg tcttatattc ttgggaattt 54240
    tcccacttca tcataaaatg accttccttg tctcatttaa tgtgttcaaa ctttgccctg 54300
    aatttaactt tgtctgatat tttaccatcc tgctgaattt tgtttgttac cccaaacaac 54360
    ctttgctgtt ttcgtctttt ctgaaccctt tattttaggt aatcccttga attagagcac 54420
    taagttttgc tttgtgatta aatctgaaaa tctttatctt gccatagatg agttgagccc 54480
    tattcatgtg acagctatat tatgctgttt catagccctt ttggtccttt tttcactctt 54540
    gcattgcata ttttgtgttt attgtgtttt gtgtttcttc tgataatttg gaaggtttgt 54600
    atttttattc agggagttgc cttataatca tactccgcaa tacacatcgt cctcagtttc 54660
    ttcagactgt ctgttaactc cctattctga ataaaaatga cattgtaatt tccctctttt 54720
    ttctttaccc cttttcttct cctcacctaa tgtaaatgat tttatccttc tttagtattt 54780
    gcttttttaa ttaactacat ttataaatat ctttatcact tgatttttaa atcagctttg 54840
    aatgagatat ttggattcct agatataaaa gatgttaatt ataccatttc cacgttagta 54900
    ggtttataaa atcatacatt ctgctgtgta accataatcc cacgtttgtt ttagttccac 54960
    tcctacagtt aaaagattca gaagtattat taacagttat tttgccatag ttttttcccc 55020
    aacccatttt gtggtaagtt atgatcctgc tttagtttct taagaataat ttatagagca 55080
    gagtgtggtg gctcacgttt gtaatcccag cactttggga gacaagaggt agaaggatcg 55140
    cttgaagcca gcagttcaag accaccctga gcaacatagt gagaccttgt ctctacaaaa 55200
    aattttaaaa tttagccaga cgtagtggcg tgtgcctata gtcccagcta ctcaggaggc 55260
    tgaggcaaga ggattgctag agcccagaag tttgaggctg cagtgacctc tgattgtgcc 55320
    actgcacccc agtctgggca agaaagtgag aacctatctc tttaaaataa caataataac 55380
    ttatgaaaat tatattccct gagtttttca tgtttaaaaa tatttgttgc ctttatcctg 55440
    taaaagtttg agtataaatt cttgggttat actttattta ttgaagaatg tataagtatt 55500
    gtcttctaga attgagtgtt gctgtaatga aaccagaagt cagcctggtt tatttttcct 55560
    cagaaatgag gtaattgccg gccggacacc gtggctcatg cctgtaatcc caacactttg 55620
    ggaggccgag acaggtggat cacgaggtca ggagattgag accatcctgg ctaacatggt 55680
    gaaaccccgg ctctactaaa agtacaaaaa gttagctggg catggtggtg gacgcctgta 55740
    atcccagcta cccgggaggc tgaggcagga gaatggcgtg aacctgggag gaggagcttg 55800
    cagagagctg agatcgcgcc actgcactcc agcctgggcg acagagtgag actccgtctc 55860
    aaaaaaacaa aaaaaaaaca aagaagtgaa gtaattgcca tgatgctcca agaattatct 55920
    ctttgtctat gaaatccaga aatctcactg ttatacattt tggaattatt attctgggcc 55980
    aatatttcct gggacacaat agattgactc tatagattta attttttttt tttttttgag 56040
    acagagtctc actgcaatct cagcttactg caacctctgc ctcacgggtt caagcaattc 56100
    tcctgcctca gcctcccaag tagctgggac tacaggcgcg tggcaccatg cctggctaat 56160
    ttttgtcttt ttagtagaga cagggtttca ccatgttggc caggctggtc ttgaacgcct 56220
    aacctcaagt gatccacctg cctcagcctc ccaaagtgct gggattacag gcgtgagcca 56280
    ccatgcccag cctcaattcc tctttctatc tggtaatttt tctgaagttg aaaacatttg 56340
    ttctaatacg ttatttcagt gttcttctaa gatgtgtaaa gcaccctatt cccaggtcag 56400
    cccccatctt gctagtgagc tcggctggtt cttcacaaga gctctggttt tctcctgctt 56460
    aatctcaagt acctctgtca gcctccacct ggtttatgat ttggagtttt ttggtttttg 56520
    ttttttgttt ttgacagagt cttactctgt cacccaggct ggagagcagt ggcataatct 56580
    cagctcactg caacctctgt ctcccaggtt tgagcgattc tcctgcctca gcctactgag 56640
    tagctgggat tacaggcgcg tgccaccaca cccggctaat ttttgtattt ttagtagaga 56700
    tggggtttca ccatgttggc cagggtggtc ttgaactcct gacctcaggt aatccacctg 56760
    cctcagcctc ccaaagtgct gagattacag gcgtgagcca ccgcgcctgg catggtttgg 56820
    agttttaatc tgtagtttta ataaagatag tgcttatgtt tgtgtttctt atatttcttg 56880
    gtactcttgg gtaatttgta agatccccat atctacacaa gaagtccatt ttcaattctt 56940
    ttcttcagac tgtttatttt attttatttt attttatttt tatgtttgag atggagtctc 57000
    gctgtgtcac ttctggaggc tggagtgcag tggcgcgatc tcaggtcact gcaacctccg 57060
    tctcccgggt tcaagcaatt ctcctgcctc agcctcccga gtagctggga ttacaggcac 57120
    ctgccacttt ttaatttttt tagagacaga gtctcgcttt gttgaccagg ctggagtgcg 57180
    gtggtgcaat catggctgac tataacctcc aaatcctggg ctcaagtgat cctcctgcct 57240
    cagcctcctg agtagctggg actacaggca catgccacca tgcccagtta attttaattt 57300
    ttttgtagag acagggtctc catatgttgc ccaggctggc ctcctactcc tggcctcaag 57360
    taatcctcct acctcagcct cccaaattac taggattata agcatgagcc accatgccca 57420
    gccttgttct actactttaa tttcatatgt taggtgacca tgtaattgat catccaaacc 57480
    aggatactgt aagaatgaaa gaggctgaca gtagtatgat gctgggacta gcattgtgca 57540
    ctgagattat ttctgggaaa gcaggagata cggtcaccct acttatagtg tgcttgtctt 57600
    tggattgttg aatttggagt ttctatttgc aggcttattt caactgggca gccttgatcc 57660
    gccctgccca gcaatgctac cgttctctcc accgggtctc tgggacccct tcagtcacta 57720
    tacttagctc agttccccac cctcccactc cctaaaagcg taaccaggaa tcctgcctca 57780
    ggtctactgc cgtcttccgt gggctgtttc agttcctatt acccagagtc aaactcccag 57840
    cattccctac ctgattccag acttggagtc cagagcttta acctcttcag gccaactccc 57900
    cactttgcat ttctgtccct atatcttagt ccatggagat acatttcatg tctttgagtc 57960
    tacttacaaa gtaaattttg ctgtttttta attttttttt tgagatggag tcttgccctg 58020
    tcacccaggc tgtggtgcaa tgacgccatc tcggctcact gcaacctccg cctcctgggt 58080
    tcaagcgatt catctgcctc agcctcccaa gtagctgtga ttacagacag gcaccaccac 58140
    gcccagctaa ttttttttat cttttagtag agacagggtt tcaccatgtt ggccaggctg 58200
    gtcttgaatt cctgacctcg tgatctgccc atctcggcct cccaaagtgc tgagattaca 58260
    ggcgtgagcc actgtgccca gccaattttg ctttttttat atttcattgc tatatgttta 58320
    gaggataagt ttacagtgct atatgcattc ccaaatatta gaccaaaaaa atctccaaaa 58380
    aattagaaag aaaatccaaa aaatctcaaa aaataccaaa aagcaacaat ctcacagacc 58440
    atactcactg acccccaata aaataaaatt agaaattaac cacaacttaa caaaataaag 58500
    tactcaagtc agagaggaaa gaggaaataa acatcaaaat tacaaagtct aggcggtggc 58560
    tcacgcctgt aatcccagca ctttgggagg ccaaggcggg cagatcacaa ggtcaggaat 58620
    tcgagaccag cctggccaat atggtgaaac cccgtttcca ctaaaaatac aaaaattagc 58680
    caggcatagt gatgtgtgcc tgtaatccag ccacttggga ggctgaggca ggagaatcac 58740
    tgaacccagg gagacgaaga ttgcagtgag ccaaaatcgt gccactgcac ttcggcctgg 58800
    gtgacaaagc gagactccat ctcaaaaaaa aaaaaattac aaactcttta gatagaaatt 58860
    ttggtgtttt tttttgagac ggagtctcac tctgtcgcag aggctggagt gcagtgggac 58920
    tatgtcagct caccgcaacc tccatctcct ggattcaagc aattctcctg tctcagcctc 58980
    ccaagtagct aggattacag gcgcccacca ccagacccag ctagttttta tatttttagt 59040
    agagatggtg tttcaccatg ttggccaggc tggtctcaaa ctcctgacct caagtgatcc 59100
    acctgcttca gcctcccaaa gtgctcagat tacaggcgtg agccaccgca ccccacctag 59160
    atagaaattt caacatgagg ccgggcacaa tggctcacgc ctgtaatctc agcacttcag 59220
    gaggctgagg cgtgggagga tcacttgggc ccaggagttc aggaccagca tgggtgacag 59280
    agacagaccc tgtctctatt tatttgaaaa aaaaaaaaaa aaagagagag agaaagaaat 59340
    ttcaacatga aaagtatctc tcaaaccctt cgagatgttg gcaaaaagcg actcaaagga 59400
    aaatgtatta ctgtgtgtga atttgcttga aaataagaaa gaggccgggt gtggtggcta 59460
    acacctgtaa tcccaacact ctgggagtcc gaatcaagtg gatcatgagg tcaggagatc 59520
    gagaccatcc tggctaacat ggtgaaaccc tgtctctact aaaaatacaa aaaattagct 59580
    aggcgcggtg gctcatgcct gtaatcccag cactttggga ggctgaggca ggtggatcac 59640
    ctgaggtcag gggtttgaga ccagcctggc ctacatggtg aaacctcgtc tcttctacaa 59700
    atacaaaaat tagctgggcg tggtggtggg tgcctgtaat cccagctact cagaggctga 59760
    ggcaggagaa tcgcttgaac ccgggaggcg gaggttgcgg tgagccgaga tcgcaccact 59820
    acactccagc ctgggcaaca gcctgggtga cacagtgaga ctccatctca aaaaatacaa 59880
    aaaattagct gggtgtggtg gcctgcgcct gtagtcccag ctacccggga ggctgaggca 59940
    ggagaatgga gtgaacctgg gaggaggagc ttgcagtgag ccgagatccc accactgcac 60000
    tccagcctgg gcgacagagc aagactcttg tctcaaaaaa aagaaaaaaa aaggaaaaaa 60060
    gaaccctgat aataaagaaa ccaaatgttc aactctcaaa gctcggacac tttaaagaaa 60120
    taattaataa aggcagaagt taaagggagg atgataaagc aatttttttt gttggttttt 60180
    ttgagatgga gtcttgctct gtcacccagg ctggagtgca gtgatgcgat cttggctcac 60240
    tgcaacctct gcctcccggg ttcaagcaat tctcctgcct cagcctcctg agtagctggt 60300
    actacaggtg cgcgccacct ggcccagcta atttttgtat ttttattaga gacggggttt 60360
    caccatattt gttaggctgg tctcaaactc ctgatctcag gtaatctgcc cacctcggcc 60420
    tctcaaagtg ctgggattac aggcaggcgc caccgcgcct ggcctaaagc aaaatattgg 60480
    ttctgtgcaa aaggtcaata aaaagagcaa acgtttacaa actggagcca gcacccattc 60540
    agctcagtgt gtctggagaa aaaacaatct cgcttcagaa ttcatgatta cgcagccctt 60600
    tttgcttcct aaaaatccta ctatgttgct gttgaccatt ctctctcttt ctctctctct 60660
    tgctttctct ccagaaaagc tattcagaca ttctcctctt tcctcaaacc tccaacactt 60720
    cctcctccat ccttagcctc agctgctgac ctcacttcta atcattgaga aaccaggaga 60780
    agcatttaag agtgaacctc cgcctccccg cacgggcaaa accacccacc cacagaattg 60840
    tgccccaatt ctgcgtcctc tcctctcacc atggatggac ggtccaggct ccgagccaaa 60900
    gccaggcctc ccctggagct ctggatccac cacctgcagc ttctcaggca gggccccagc 60960
    agctcccctg ctcccttgta ccatcaatcc ctcccctcac tgggtcactc ccaacaatat 61020
    atatatttag tgatgtttct cccatgtggt aaaatcactt agcctctctc ctcccccagc 61080
    tactatccta tttgtttctt tccattctct gcaaaacttc tcaaagcatt gtgtctatgt 61140
    gctgactcca tttatcttct cccgttctct gctgagtcct tcccacagac tctcacccca 61200
    gttactccat gaaatgacct ctgcactgcc acatccaatg gtgaatgttc agttcttaat 61260
    tttattcagt ctttcagcag catttgacct ggccgatcac tccctcttct taaaaatact 61320
    tttctcagcc aggcgtgatg gctcacacct gtaatcccaa cactttggga ggccaaggcg 61380
    ggaggatcat gagagcccag gagttcaaga tcagcctggg caacatggca agaccctatc 61440
    tctacaaaaa ctaaaaagta gccagtgtga tggcatgcac ctgtagtccc atctacttag 61500
    gaggctgagg cagtaggatg acttgagcct gggaaatcaa ggctgcagtg agccatgatt 61560
    gcaccactgc actccagcct gagtgacagc gagaccctgt ctcaaaaaga caaaatagga 61620
    aacttttctc agcatattcc tctgattctc ctgctgcttc tgtctgcaca gattcagtct 61680
    cctttgccgg ttcttcctca tcctcctgat ctcttgacct tgaagtgccc cagagtacag 61740
    tctttttttt tttttttgag acgcagtctc gtctgtcacc caagctggag tgcaatggcg 61800
    aggtctcagc tcatgcaacc tctgcctcct gggttcaagc gattctcctg cctcagcctc 61860
    ccaagtagcc aggactacag gcacatgcca ccatgcccag caaattgttg tatttttagt 61920
    agagacaggg ttttactata ttggccacgc tggtctcaaa ctcctgaact cgtgaaccac 61980
    ccgcctcggc ctcccaaagt gctgagatta caggcatgag ccaccacacc cggcccagag 62040
    tacagtcttt agacggcctc tctacctata cttgctcccc tcataaactc ctcctgcctc 62100
    atggctttaa ataccatcgg tagactgatg actcccatat ttctcttttt tttttggaga 62160
    cggagtctcg ctcagtcccc caggctggag tgcagtggcg cgatctcggc tcactgcaag 62220
    ctccacctgc caagttcaca ccattctcct acctcagcct ctccagtagc tgggactaca 62280
    ggcacccgcc accacgcctg gctaattttt ttgtattttt agtagagatg gggtttcacc 62340
    atgttagcca ggatggtctc gatctcctga cctcgtgatc cgcccatctc ggcctcccaa 62400
    agtgctggga ttataggtgt gagccaccgt gcccagccga tgactcccat atttctatct 62460
    cttgctgtgt gggagttctc ctcagaactc catactcata aatccaactc tcataaatag 62520
    tatctcaaat gggcaatatg ctcaaaagtc aattcctact tttctcccta aacttgcttt 62580
    cctgcagtct ccaccatctt aatgtccaat ctaacattag gaggcaaaaa ctttgaagtc 62640
    attcttgact cttctctatt acacacccta tccaatcttt ctgcagatcc agtcgacccc 62700
    caaatccagt tagctctcat catctcccct gttaccccct ggtccaggcc atcttcctct 62760
    ctcacctgaa tcactgcagc attctcctca ctggtctctt tggttctgtt ttcactccac 62820
    cttagcatag tctccacaga gcagtcagag ggatcctttt aaagtgtaat tcccatcctg 62880
    tccctgctct gctcaaaacc ctgtcgtgat tcccgtttta atctgtcaga ttaaaagcca 62940
    gagtctttcc agtgacctac atgatctgcc tattatcacc tcccacttct ttccccttgc 63000
    tcactccact ccagctctgc agctgtcctt tctgtttcct gaacagccca gattttgctt 63060
    ctttagaacc tttgtatttg ctgtcccctc tgtctggaat gtttttccag gaagtcacct 63120
    ggctctctcc tgcacttcct tcctgaccac catgtttaaa aatcactcaa acacacttca 63180
    ggccggacat ggtggctcac gcctgtaatc ccagcacttt gggaggccaa ggtgggtgga 63240
    tcacctgagg tcaggagttc gagaccagcc tggccaacat ggtgaaactt cgtctctact 63300
    acaaatacaa atagtagcca ggtgtagtgg cacacacctg taatctcagc tactcaggag 63360
    gctgaggcag gagaatcgct tgaacccaga aggcagagga ggtgcagtga gccaagatca 63420
    cgccacaaca ccccagcctg ggtgacagag caagacccca tctcaaaaaa aaaaaaagaa 63480
    aaaaaaatca cacaaacaca cttctcttca tattcctttt ccaagtttta tttttctcca 63540
    gaatacttta cattgtttta atggaagttc tccgtttccc cccaactaga atggatactt 63600
    cctgcaggta ggcactctag tcctcccatc caagtactaa ccaggctcaa ccctgcttag 63660
    cttctgagag caggggagat caggcctgtt cagggtggta tggcccagga attttgattc 63720
    tgttttattc attgctgttc tgttgattct cttttgttcc tcctcctagt gctgagaaca 63780
    ctacttgtac ataataagca ttcaataaat atttgttgaa tgaatgactt gttgaatgaa 63840
    ttaatctcag aaatgcagga ctggttctac attagaaaat ttttcaaggt cattctctgt 63900
    tgtcgtaaca cattaagaga ggaaaatttt gtactctaaa tcatttgata aaatacatac 63960
    tgatttctgt tttcaaaaac tcttagtggc tgggcgaggt ggctcacatc tataatccca 64020
    gcattttggg aggacgaggt gggcggatca cttgaggtca ggagtttgag accagcctgg 64080
    ccatcatggt gaaaccctat ctctactgaa aatagaaaaa ttagccgggt gtggtggcgc 64140
    atgcctgtag tcccagctac ctgggaggct gaggcaggag aatggcttga acccgggagg 64200
    cggaggttgc agtgagccaa gatcatgcca ttgcactcca gcctgggtaa cagagtgaga 64260
    ctccatctca aaagaaaact cttagtgagt ttaggaatcc aaggaagacc ctcaaactaa 64320
    atagataatc tagctaccag aagccttcag taaaccttaa cactccatgg tgaaacatta 64380
    gaaacattcc tactaaaaga caggctaaga atgcctgcaa tcttcacggc tagtccaaga 64440
    agtcaaaaag aagaaatgag cgctgattta aaaaaataaa caaacaaaaa actaccgatg 64500
    cagaggctgg cagcaaggac tgaaggactg tacagtactt gcctggagca ggcggatggc 64560
    cacacccctg cgaagcctgc tcagctggct gggggacgct ccagtgtgtg agtggcagga 64620
    tgcagggtac ttcctctgcc agggagttgc actggggaga tcctccccca ctcacacttt 64680
    ggcagctggg gctttggaat gtgacttagc ttctgtcaaa gggtcaatcc accctttgat 64740
    atatgatgca aaggcgaaca tatgatgcaa aggtgagaga acagcccaaa ttaggacttt 64800
    taccacagct gtggaggtgg acagcgacag tggtgggccc tggccagact tttcatgctc 64860
    aaaggtggtg gttgttcttc ctacttcttg tccctccagg gcttcctttg cctgtgtgct 64920
    gaacctgctt cttttaattt tttttaactt ttttaaattt ttaattgttt taattaaaac 64980
    aaattttgaa aactgtctga acctgctttt gaaccctgct atgatttgaa tgtttgtccc 65040
    ctgccaaact gattttgaaa cttaatctcc aaagtggcaa tattgagatg gggctttaag 65100
    cagtgactgg atcatgagag ctctgacctc atgagtggat taatggatta atgagttgtc 65160
    atgggagtgg catcagtggc tttataagag gaagaattaa gacctgagct agcatggtcg 65220
    ccccttcacc atttgatatc ttacactgcc taggggctct gcagagagtc cccaccaaca 65280
    agaaggctct caccagatac agctcctcaa ccttgtactt ctcagcctct gtaactgtaa 65340
    gaaataaatg ccttttcttt atgaattacc cagtttcaga tattctgtta taaacaatag 65400
    aaaacgaact aaggcaaact ctcatgattc tactgccatg ccattccaat aaactccctt 65460
    tatgcttaag agagccagag ttggccaggc gtggtgactc acgcctgtaa ttccagcact 65520
    ttgggaggcc gaggcaggtg gatcacaagg tcaggagatc gagaccatcc tggctaacac 65580
    ggtgaaaccc cgtctctact aaaaatacaa aaaaattagc tgggcgtggt agtgggtgcc 65640
    tgtagtccca gctactcggg aggctgaagc aggaggagaa tggcgtggac ccaggaggcg 65700
    gagcttgcag tgagtcgaga tcgtgccact gcactccagc ctgggtgaca gaatgagact 65760
    ccgtctcaaa aaaaaagaga gccagagttt atttctgttg cttgcaacca agaaatctgg 65820
    ctggtgcact gaagtttcca taaataatag caatttaaag actctttcca agccaggcaa 65880
    tgcctagcct tgtgtagtcc ttgtggtaat acattcattc attcatttgt tcaaccaact 65940
    gtgctccaga gactaagaat acaaaaatgg gggccgggtg tggtggctca cacctataat 66000
    cctagcactt tgggaggccg aggcaggtag atcacctgag gtcaggagtt cgagaccaac 66060
    ctggccaaaa tggtgaaacc cctactctac taaaaataca aaaaattagc tgggggtggt 66120
    ggcggacacc tgtaatccca gctactcgtg agactgaggc aggagaatca cttgaacccg 66180
    ggaggcagag gttgcagtga gccgagatcg caccactgca ctccagcctg ggcaacaaga 66240
    gcgaaactcc acctcgaaaa aaaaaaaaaa aaaaaaagag ggccggggct gggcgcagtg 66300
    gctcacgcct gtaatcccag cactctggga ggccaaggca ggagaattac gaggtcagca 66360
    gatcgagacc agcctgacca acatggtgaa accccatctc tactaaaaat acaaaaatta 66420
    tccgggcgtg gtggcgcaca cctctagtcc cagctacttg ggaggctgag gcaggagaat 66480
    cgcttgaacc cgggaggcag aggttgcagt gagccgaaat catgccactg cactccagcc 66540
    tgggtgacag agtgagactc cgtctcaaaa aaaaaataaa aaaaaaaaaa gaattcaaaa 66600
    attgtagagt tatagtgtgc ttctagttta gttgagagga catctgtcct tcaaggaagg 66660
    ctagaatcta taccctgagt ccttactgaa atcaatccag cagtcaaaac atgggaccaa 66720
    cgatcacagc agtaagatag gaagagcacc tttgtacatt tagctcatgt tgagataagc 66780
    cactgacaga gctgaaggaa gctcacagtt ctgggttcca tcctttggca tttaaaaaga 66840
    aaagtgctaa gaaaattcgg ttggtcacgg tggctcacgc ctgtaatccc aacactttga 66900
    gaggccaagg caggcagatc acgaggtcag gagttcgaaa ccagcctggc caacatggtg 66960
    aaaccccgtc tctactaaaa acagaaaaat tagccgggca tggtggcgca tgcctataat 67020
    cccagctact caggaggctg aggcaggaga attgcttgaa cccgggaggg ggaggttgca 67080
    gcgagtgaga gcaggccact gcactccagc ctgggagaca gagcaagact ctgtctcaaa 67140
    aaaaaaaaag aaaaaaagaa agaaaggaaa aaaagaaaga aaaaaaaaga aaaaagaaaa 67200
    ttcaggccag gccaggcctg gtggctcaca cctgtaatcc caacactttg ggaggctgaa 67260
    gcgagacggt gccttagccc aggagtttga gaccagcctg agcaacatag cgagaccctg 67320
    tctctataaa aaaaaatttt tttttggcca gacgcagtgg ctcacgcctg taatcccagc 67380
    actttgggag gccgaggcag gtggatcacg aggtcaggag atggagacca tcctggctaa 67440
    cacggtgaaa ccccatctct actaaaaaat acaaaaaatt aaccgggcgt ggtggcgggc 67500
    gcctgtagtc ccagctactc gggaggctga ggcaggagaa tggcgtgaac ccgggaggcg 67560
    gagcttgcag tgagccgaga ttgcgccact gcactccaga ctgggagaga gtgagactcc 67620
    gtctcaaaaa aaaaaaaaaa aaaaaaaaat taattgtcag gtgtgctggc atgcagctgt 67680
    agtcctagct actcgggagg ctgaggtaag aagatcgctt gagcccagga gttcaaggct 67740
    gcagtaatag tgcctctcac tctaccctgg gtgacaatga gaccctctct caaaaagaaa 67800
    gaaaaaaggg aaagaagaaa agaaagaaag aaagagaaga aaggaaggaa gaaagaaaga 67860
    aaaagaaaag gaaggaagga agaagaaaaa aaaagaaaga aagaaaagag agagaagttc 67920
    aaagaccaaa gggtcaggat cccaaaatag tttttatgtt ttatttattt atttacttat 67980
    ttatttttga gacagtatgg ctctgtcgcc caggctggag tgcagtgatg cgattgcggc 68040
    tcactgcagc ctccaaactg ggctcaggtg gccctcccac ctcagcctcc cgagtagctg 68100
    ggaccacagg cgcgtgccac catgcccagc taatttttta attctttgta gagatgaggt 68160
    ctctatatgc tgcccaggct ggtctcgagc tcctgggctt aagccatcca cccgcctggg 68220
    cctcccaaag tgctgggatt acagaagtga gccaccgcgc ctaatcgggt ggtttgtttg 68280
    tttattgacg gggtctcgct gctgcccagg ctggagtgcc agtggctgtt cacaggtgca 68340
    gtcctggagc attgcatcag ctcttgggct ctagcgatcc tccagagtag ctgcagctgg 68400
    gattccaggc gcgccaccgc gcggggctca gaatgggttt ttatattgag ggttatgctg 68460
    ccacctagag gatatatgta gtaccgaact gtgtgcgcag ggaggctgag gttgcagtga 68520
    gccaagatga tgccagggca ctccagcgtg ggtgacagag caagatttca tctcaaaaaa 68580
    aaaaaaaaaa aaaaaaaaaa aagaattgaa agtaaggtct tgaagagata tttgtgcctg 68640
    tatggtcata gcagtattaa ctttgaccca ctagctaaaa cacaaaagca acatgtgtct 68700
    gtcagcaggt gaacggataa acaaaatgtg gtatatatgt acaattgaat attattcagc 68760
    ctttaaaaag gaataaaagg ctggatgcgg gggctcacgc ctgtaatcct aacactttgg 68820
    gagactgagg tgggtggatc acccgaggtt aggagtttga gaacagcctg gccaacatgg 68880
    tgaaacttca tctctactaa aaatactaaa attagccggg catggtggca cttgtctgta 68940
    atccaagcta ctggggaggc taaggcagga gaattgcttg aactcaggag ccggaggttg 69000
    cagtgagcta agatggcacc actgcactcc agcctgggca acagagtgag actccatctc 69060
    aaaacaaaca aacaaaaaat tattatttcc aaagaaacaa gaccctgggt ccatttccca 69120
    gcccacacct gatgttgact cacaacacac agcctggttt gctatgagcc tgcttcattt 69180
    aattgtcacc ttaacttcac atcaccctca agtcctggaa taactctttg ctgacctttg 69240
    tgtgctgagc catctccatg tcgctcaacg tgcagtccct ctcactgcac tgagtcaata 69300
    gccagacgtg gtctgactgc agggtcatcc ttggtggctt aggctgactc gggcatagca 69360
    gggtgctctg agacctcacc gcatataggc tttgccccca ataaactcta tataatattc 69420
    atattatgtg gtctgggtgt gtgtagcttt gcactgtctt ctcgtgacag tgccctcaac 69480
    ctctttccca ggatttcctc ctctacctcc tcaagtccca ctgctctgca aagaccaaaa 69540
    gctgcagagt cccagctccc tcctttacac cccacgacgc agcctcctct ctcagaaccc 69600
    tttaaacaga gtcttttact gcagatccca agaacagcca cacccctctc tcccacccac 69660
    tccagacaca cccaggtaat tatagcaccc agggtaacta tgtagatgga gtccctggaa 69720
    catgtggata gtgccccctg ggagtatgca aaagcaacat tgctggcacc tgcagagaac 69780
    agggtgacat ccaggaatca gagcatgggc ctctgggagg tagggatgtg gccaggcagg 69840
    ctgccaaaaa ttggtagagc aaggccacag gatctttctg accttccttc caaacagagg 69900
    ctcctgtact ggtgatccct gtgttgattg accactccct tcctgggggt cgtggtctct 69960
    gtcccagttg cccggacttc tgtgagtgtc ctactgaggt ccttttcatg agaagcatgc 70020
    tgtccttcca cctgctggga gcaagagtga caacttcaat actataatag cagtggcata 70080
    cagagaagaa gaaagatgaa gtggcaagaa aaacaggctt ccaagcagga gtttttctat 70140
    aaaaacaaaa acgtttacaa gcaaactttt tataaagggc tagatagtaa atattttagg 70200
    ctttgagagc cacatagact tgtttgcagg gactcaatgt cgctattgta gtttgaaagc 70260
    agccatcagg gttatgtaaa tgagtgagtc tgattttgtt tcagcaaaat tttatttacc 70320
    aaaacagaca atgagtgggc tggatttggc ccatgatcct tagtttgcca actcctgctt 70380
    tgggctcacc cagatctgat tttgaattct ggctctgcta ctggttagct gcaggagctt 70440
    ggaaggctct ctgagcctgt ttcctcatct gtaaaattaa agcaataatt tctaacactc 70500
    aagagtgtta cctcacgcct gtaatcccag cactttggag gctgaggcag gcggatcacc 70560
    tgaggtcaga agttcaagac cagcgtggcc aacgtggcaa aaccctgtct ctactaaaaa 70620
    atacaaaaag tagccgggca tggtggcgcg catctgtaat cccagctact tgggaggctg 70680
    aggcagggat actgctagaa cctgggaggt ggagcgtgca gtgagtggag atcacacctc 70740
    cacactccag cctggccgac agagcgagac tccatctcaa aaaaaaaaaa aaaaagagtg 70800
    ttagaaggtt ttgagataat gaataaaaga tgccttgtgt atactaagta ttcaacaact 70860
    gatagctgca ttggtctaat tataacagtt tagaagcgat tgagtcaaca aatgctggat 70920
    ttgtcaggga ggacttccta tcaggaggta gatcttgggc tgagtcctga agcaaagata 70980
    ggcattggat agaggagttg agagaacacc ctaggactgt tattattatt attcgacacg 71040
    gagtctcttg ctctgtcacc caggctggag tgcagtggcg cgatctcggc tcactgcaac 71100
    ctctgcctcc caggttcaag cgattctcct gcctcctaag tagctgagac tacaggtgtg 71160
    tgccaccaca cccggctaat ttttatattt ttagtagaga cagagtttca ccatgttggc 71220
    catgctggtc tcgaactcct gacttcaggt gatccacccg cctcagcctc ccaaagtgct 71280
    ggaataacag atgtgagcca ccgcacccag cccagaacca tttttcaatc cttggctctg 71340
    ccttttatta gctgcaagat ctcaggcaat ttatttaacc tctccaaaga ctcattttct 71400
    cattcacaaa atgaggcaaa taataatatc tactatccca ggttgtcatg agaattaaat 71460
    gcaacatgac atttaatgaa atgagaagtc ccttggacat taactggcta aagtatgtgc 71520
    tcgacaagga tatcatttta ggtggatact tagcatctca gaactgatgc tcacaatgga 71580
    atatcattga aacgcattaa aattcatttt aaatgattgt aggtagtgag gcaattgaaa 71640
    gaagaagaca agaggactga ttataatgct tcaggctcac tagtctcctt ttaggaggga 71700
    aaaacaattt caagttaaat tttaggctct agatttttac ccctgctgct cattagaatc 71760
    acccagattg atgaaatcag agcccatctg aggctgtgtt tttcatctcc agaatgagag 71820
    ctgttgtggg gattaagttt ttgaaaaagt acatctaaca ggtgatcgaa aatgatagtg 71880
    atattattgc agtgatggtc attattgttg ttattattat actgaaagag gcttcagttt 71940
    tctgatccat aaagtgaggg aattgcatga gaccattgct aagattcctt ctagctctgt 72000
    ttttttgttt ttgtttttta gacagagtct ctgtcgccca ggctggagtg caatggcatg 72060
    atcttggctc actgcaacct ccgcctcccg ggttcaaatg atcctcctgt ctcagcctcc 72120
    gaagtagctg ggactacagg cacacaccac catgcccagc taacttttat atttttaata 72180
    gaggtggggt ttcaccatat tggtcaggct ggtctcaaac tcctgacctc aggtgatcca 72240
    cccgcctcgg cctcccaaca tgctgggatt acaggcatga gccactgtgc ccaacccctt 72300
    ctagctttct tgatcactga ttctagggtt ctctgctgaa atatatttga gacatcctgg 72360
    ataaaagatc atgcaagagc tcccaatatg gtattaataa ttgattctgg aggcttagct 72420
    actcctgatg gattagacat gactcaactg cctctcttat gtgtacaaca caacaacaca 72480
    accaagaaag gttattctgg cattccattt attcagttta tttacagccc ttacttccag 72540
    cagcacgtta aagatatggc cagggccggg tgcagtggct caagtctgta atcccaggac 72600
    tttgggaggc caaggtgggc ggatcacaag gtcaggagtt tgagaatctg gcaattcttc 72660
    agacttagaa gcaaccagct cgataacaca gtcttgtgtg ggctctccct ctgtccctcc 72720
    ctcgcttccc tcatttctca tccctgcccc tgagactgtg caccttcaca tagccctgcc 72780
    atgagacctt catctcaggc tttgctttct ggggtaactg aggctaaaca ctgagtggcc 72840
    ctaaaagagg attgggattt ggaagttaga ttattcacca gagaacagac tttgctgatg 72900
    atcaggccca ggttgtaatt gttgaaaaaa agagaggatg catagtctta tctcatctcc 72960
    tagtcaaagt caacaccatg ataaataaga gtcaaatcct gagatgtgaa ttggggacat 73020
    ttgagtggtt aaccctgaga agcttgcacc ttcagacccc tcaatacccc tgctccccag 73080
    agaaggctgg acattgacct cagcacaggc aggagccctg caagatgcca tttgtcctac 73140
    taaagatgga cccctccact ctgtttctag gtaaataacc aaagtcaagt ctccacacag 73200
    cctgagcaag aaagtcagag cctgctacag gagaaaatac cacactggcc aaaggattca 73260
    ctagccctgg ccactgtgtg tgggaggaac cagggaatca tgtgtgggag tcaatgttga 73320
    agctgttgga ctgggggtgg ggtggaatat aagcctggcc ctggggagtt tttcccgttt 73380
    gagggccttt acccacaact caagatccag tgctatagca ggagatccca gagctagtcc 73440
    taacagatgg tcaggattga acttggccta gagtaaaatg aggaggatag tgccagaact 73500
    ttctcaacat actattgagg aagaggtcag aaggcttaag gaggtagtgt aactggaaag 73560
    gggtcctgat ccagacccca ggagagggtt cttggacctt gcataagaaa gagttcgaga 73620
    cgagtccacc cagtaaagtg aaagcaattt tattaaagaa gaaacagaaa aatggctact 73680
    ccatagagca gcgacatggg ctgcttaact gagtgttctt atgattattt cttgattcta 73740
    tgctaaacaa agggtggatt atttgtgagg tttccaggaa aggggcaggg atttcccaga 73800
    actgatggat ccccccactt ttagaccata tagagtaact tcctgacgtt gccatggcgt 73860
    ttgtaaactg tcatggccct ggagggaatg tcttttagca tgttaatgta ttataatgtg 73920
    tataatgagc agtgaggacg gccagaggtc gctttcatca ccatcttggt tttggtgggt 73980
    tttggccggc ttctttatca catcctgttt tatgagcagg gtctttatga cctataactt 74040
    ctcctgccga cctcctatct cctcctgtga ctaagaatgc agcctagcag gtctcagcct 74100
    cattttacca tggagtcgct ctgattccaa tgcctctgac agcaggaatg ttggaattga 74160
    attactatgc aagacctgag aagccattgg aggacacagc cttcattagg acactggcat 74220
    ctgtgacagg ctgggtggtg gtaattgtct gttggccagt gtggactgtg ggagatgcta 74280
    ctactgtaag atatgacaag gtttctcttc aaacaggctg atccgcttct tattctctaa 74340
    ttccaagtac caccccccgc ctttcttctc cttttccttc tttctgattt tactacatgc 74400
    ccaggcatgc tacggcccca gctcacattc ctttccttat ttaaaaatgg actggggctg 74460
    ggcgcggtgg ctcatgcctg taatcccagc actttgggag gccgaggcgg gcggatcatg 74520
    aggtcaggag atcgagacca tcctggctaa cacggtgaaa ccccgtctct actaaaaatg 74580
    caaaaacatt agccaggcgt ggttgcaggt gcctgcagtc ccagcggctc aggaggctga 74640
    ggcaggagaa tggcgtgaac ctgggaggtg gaggttgcaa tgagccgaga ttgtgccact 74700
    gcactccagc ctgggtgaca gagcgagact ccgtctcaaa aaaaaaaaaa aaaaaaaaaa 74760
    tagctgggca tggtggcgcg tgcctgtaat accagctact ctggaggctg aggcaagaga 74820
    atcgcttgaa cccagtaggc ggaagttgca gtgagccgag atcttgacac tgcactccag 74880
    cctggtgaca gagtgagact ctgtctcaaa aaaaaaaaaa agaaaaaaaa agacagaaag 74940
    aaagagcaca gacagagtca caggtatttg cagtaggaag ctgtcaggtt agagtgcacg 75000
    gaaatagaaa gtatatttta cacttacagc acatcttcgt ttgattagcc acatttaaaa 75060
    tactgaatag caacgtgtgg ctatttagta ttcactaaaa tcttggacag tgcaagtcta 75120
    aagaatcctt gatccgtccg gcatggtggc tcacgccttt aatcccagca ctttgggagg 75180
    ccaaggtgga aggatcactt aaggtcagga gttcgagacc agcctggcca acatggtgaa 75240
    acctcgtctc tactaataat acaaaaaaaa ttagccgggc atggtggtgc atgcctgtaa 75300
    tcccaggtac ttgggaggct gaggcaggag aatagcttga atccaggagg cgctgcagtg 75360
    agccgagatc atgccatgcc actactgcac tccagcctgg gcaacagagt gagactgtct 75420
    caaaaaaaaa aaaaaaattg ttgggcgtgg tggctcacgc ctgtaatccc agcactttgg 75480
    gaggctgagg ggggtggatc acctgggttc tggagttcga gaccagcctg gccaacatgg 75540
    tgaaacccca tctctactaa aaatacaaaa attagctggg cgtggtggtg ggcacctgaa 75600
    atctcagcta ctcaggaggc tgaggcagga gaatttcttg aacccaggag gcagaggttg 75660
    cagtgagcca agatcgcgcc tctgcactcc atcctgggtg gcagagcaag actatgtctc 75720
    aaaaaaaaaa aaaaaaatac ttgattgtct ggacattctg cagaacatca tatggagaca 75780
    ctatgttgac gacatcatgc tgattgtaag caagaaatgg caagtgttcc agaaacacag 75840
    tcaagacaca tacatgccag aaggtgagat ataaactcta ctaagattca gtggcctgcc 75900
    acactggtga catttttaaa cctgctagat gtttgtgtag aaaaggattt aaccttgccc 75960
    aaagaggggt ctggcctttg tccccagcta ctggacataa tctctttaaa ctcttgaaat 76020
    atcattcctg atagaagtat ttttgttttg actaggggcc ttgggccagc cagatagcaa 76080
    caatgtgatc tgggttgggg gctttggatc aggtggcatc agtgtgacct cctgagtggc 76140
    tagagactag aatcaaccac atgggcagac aacccagctt acatgatgga attccaataa 76200
    agactttgga cacaagggct tgggtaagct ttcctggttg gcaatgctct atactgggaa 76260
    acccattctg actccatagg gagaggacaa ctggatattc tcatttggta cctccctggg 76320
    ctttgcccta tgcatttttc ccttgtctga ttattattat tattatgaga tggaatctcg 76380
    ctctgtcacc caggctggag tgcagtggaa tgatctcaac tcactgcaac ctctgcctcc 76440
    ccggttcaag cgattttcct gtctcggcct cccgagtagc tgggactaca gatgcatacc 76500
    accacacccg gctaattttt ttgtattttt agtagagacg gggtttcacg ttagccagga 76560
    tggtctcgat ctcctgacct catgttccgc ctgcctcggc ctctcaaagt gctaggaata 76620
    catgtgtgag ccaccgcgcc cagccccctt ggctgattat taaagtgtat ccttgagctg 76680
    tagtaaatta taaccgtgaa tataacagct tttagtgagt tttgtgagca cttctagcaa 76740
    attatcaaac ctaaggatag ccttggggac ccctgaactt gcagttggtg tcagaaataa 76800
    gggtgctcat gtgtgtacca tgccctctaa ttttgtagtt aattaacttt cacaacttta 76860
    ttattaccgc ttacactcaa tgtttattca catttatcca cataccactt attctagtgc 76920
    cttgcatcaa agactttcta tctcatgtac tttattctgc ttgaagtaaa tcctttagga 76980
    tattcttttt tttttttaaa ctttgcacat acatactttt attttttatt tatttttaat 77040
    tttgttattt ttgtgggtac gtagtagata tatgtattta tggagtacat gagatgtttt 77100
    gatacaggca tgcaatgtga aataagcaca tcatggagaa tggggtatcc atcctctcaa 77160
    gcaatttatc cttcaagtta caaacaatcc aattacactc tttaagttat tttaaaatgt 77220
    acatttaatt ttgtattgac tagagtcact ctgttgtgct atcaaatata attttttttt 77280
    tttttgagac agagtctcac tcagtggccc agactgaaag tgcagtggca caagctcggc 77340
    tcacttcaat ctctgcctcc ctggttcaag cgaatctcct gcctcagcct cccacatagc 77400
    tgggattaca ggcacacacc accatgccca gctaattttt atattttttt agtagagacg 77460
    ggttttcgcc atgttggcca ggctggtctt gaactcctgg cctcaaatga tctgaccacc 77520
    tcagcctccc aaagtgctag gattacaggc atgagccacc acacctggcc aaaatagaat 77580
    attctttagt gaggtctgct ggtgacaatt tttttctttt ttttgagact gagtctcgct 77640
    gttgtcagct tgggctggag tgcaatagca cgatctcagc tcactgcaac ctccacctcc 77700
    cggattccag caattctcct gcctcagcct cccaagtagc tgagagatta caggcaccca 77760
    ccaccacacg cggctaattt ttgtattttt agtagaaatg ggggttcacc gtgttggcca 77820
    ggctggtctc gaactcctga cctcaggtga tccacccacc ttggcctccc aaagtgctgg 77880
    gattacaagc atgagccacc acgcacagcc aattttttcc gtttttgtct gaaatcttat 77940
    tttgtgtcat ctttgaaata tatttttgat ggatataaaa ttgttggttg atagttatta 78000
    tcattattat tattattttg agacagggtc tcactctgtt gcctatgctg gggtgtagta 78060
    atgtgatctc ggttcactgc agacttgacc tcctagggct caggtgatct tcccacctca 78120
    gcctccctag tagctgggac tacagatgca tgccaccata cccaactaat ttttctattt 78180
    tttgtagaga tgaggctttg ccacatttcc caggctggtc tctaactcct gagctctagc 78240
    aatccaccca ccttggcctt acaaagtgct gggccatgac tagccagcag ttacttttta 78300
    tagcatattg aatatttaat atgaatcttc tggcatccac tgtaactgtt taaaaaatca 78360
    gctgtttact tggcactctt tttttttttt ttttttttga gacagagtct tgccctgtcg 78420
    cccaggctgg agtgcagtgg cgtgatcttg gctcactgca agctctgcct cccgggttca 78480
    cgccattctc ctgcctcagc ctccggagta gctgggacta aaggcgcccg ccaccacgcc 78540
    cggctgattt ttttgtattt ttcgtagagt tggggtttca ccgtgttagc caggatggtc 78600
    tcgatctcct gacctcgtga tctgtccgcc tcggcctccc aaagtgctgg gattataggc 78660
    gtgagccacc gcgcccagcc tctttttttt ttttttttag acggagtctt actctgtcat 78720
    ctaggctggt gtacagtggc gtgatctcag ctcagtgcaa cctccacctc ctgcctcagc 78780
    ctgccaaata gctgggatta caggtgcgta ccatcacgcc cggctaattt ttgtattttc 78840
    agtagagatg gggtttcacc atgttagaca ggctggtctc gaactcctgg cctcaagtga 78900
    tctgcctgcc ccagcctccc aaagattaca ggcatgagcc accgcacccg gccaagtagc 78960
    actcctttga aggtaatctg cttcccctac ccctagcaat ttttaacaat ttttcttcat 79020
    ttttatttcc tgaagttttg ttattaataa tctgtgtgca gatttctttg tatttctttt 79080
    gtttgcagtt catagtgatt cttgaattag tgtgttggtt tctgttatca ccacaggaaa 79140
    attgtcagcc gttagctttt caaatatttc cttgctaaat tctctcttct cccctttcgg 79200
    tacaattgat ttgattaaaa ctaaaaccag ggccgggtgc agtgactcat gcctgtaatc 79260
    ccaacacttt gagaggctga ggcaggtgga tcacctaagc tcaggagttc aagaccagcc 79320
    tggccaatat ggtgaaaccc cgtctctact aaaaatacaa aaattaccag gcatggtggc 79380
    acacatttgt agtcaggagg ctgaggcagg agaattgctt gaatccagga ggtggaggtt 79440
    gcagtgagct gagatcccac cactgcagtc tggcctgggc gacagagtga gatgagaatc 79500
    tgtctcgaaa aaaaaagtta tgaatgtttg ataaactata tttgttagaa tgtttgttgt 79560
    agaatactat tcattgattt ttaaacaatg ttagattaaa ccattcactg gatttgtgat 79620
    aattaactta ctgattttac ctcactgatt tgttgtaatt aatacaactg gtataaaaag 79680
    actgtgacga ggccgggcat ggtggctccc gcctataatc ccagcacttt gggaggctga 79740
    ggcaggcgga tcacctgagg tcaggagttc aagaccagcc tgaccaacat ggtgaaaccc 79800
    catctttact aaaaatacaa aattagccgg tcgtggtggt gcatgcctgt aatcccagct 79860
    cttcgggagg ctgtggcagg agaatcactt gaacccggga ggtggaggtt gcagtgagcc 79920
    gatatcgcgc cattgcactc cagcctgggc aacaagagcg aaactccgtc taaaaaaaaa 79980
    aaagaaaaaa aacacataaa acaaaacaac actgtgacgg ttcccaaaaa ttaggagcat 80040
    aattaaagga actcctgata aaaattaatt ttatcttaca tgtaaactaa aatgacttta 80100
    tgaagttaat tcagaaatac aatgcagggt attagtttgc cacagctgcg tattcagcct 80160
    aatgtaatat tcttgttatt tttaaattct tcttttaact ttactcatat gtggatcatc 80220
    aaatttcaaa agattaaatg acaatactct tagcagcaag cttccctaag catataaaca 80280
    ttttaatggg tgatgattca gaaggtaccc gaagaatatg tactgccaga tatcattcac 80340
    ccccatatac ctgcccgaca gacatcccat tttgggaccc tggataaatg tgtgggtgga 80400
    gagaaagata ggagaaagtg gtataagcaa atggctttgg agtctgattg acagcgattg 80460
    aaatcctgtc tctacctctt aacagcctca tgatcctaca taagttaccc cgatcctcag 80520
    ggccacatct gtaaattggg ggttgcgatg gcagccatct cacagggtct cttttcgggg 80580
    aagggcagga attatggatt aagtgagcta gtaattgtaa agcacttaat acaaggaggg 80640
    cgcataataa gtacttcata aataatgacg gccattatca tgactgaggt gtatgcagct 80700
    gtcggggatt acggcgactt cagaatttct ggtgggcagg gctcaaaggc agcaaatcac 80760
    actggaagtc gaggtgaggc actgcttctg cacagactgc ttagctggag agaatgagga 80820
    aggcttagag gagatttaga ggaacttaga gtcctccgcc tccaactctg tgggatctgc 80880
    tcccgtgcca gagacattca ggggatttct cgcactctcc cctcccctac gtccctcccg 80940
    ccccatccaa ctaaccacac aacacataca aaatagcccc tgcgaggttc tgcacgctgg 81000
    aagggaacag gagaagggcg ctgcgctttc ttgctgatgc cctgtacttg ggcccctggt 81060
    agacacagcc acttgtcccc tcagcctgca gagaaatccc acgtagaccg cgcccgggtc 81120
    cttggcttca gccaatctcc ctttggtggg ggtgggatgc acgatccaag gttttattgg 81180
    ctacagacag cggggtgtgg tccgccaaga acacagattg gctcccgagg gcatctcgga 81240
    tccctggtgg ggcgccgctc agcctcccgg tgcaggcccg gccgaggcca ggaggaagcg 81300
    gccagaccgc gtccattcgg cgccagctca ctccggacgt ccggagcctc tgccagcgct 81360
    gcttccgtcc agtgcgcctg gacgcgctgt ccttaactgg agaaaggctt caccttgaaa 81420
    tccaggcttc atccctagtt agcgtgtgac cttgagcagt tgactttatt tttcagtgcc 81480
    tagttttcca gataccagga ctgactccaa ggactattac tcatctggag ggtttagcac 81540
    agtaccgtcg catagtaaat ttccatgtca gttttggtta cctttcatgc acttgcaaac 81600
    atgccatgct ctgaaacgaa ataggcacat cttttttttt ttttttttta aggagtcttc 81660
    ctctcgccca ggctggagtg cagtggcgcg atcttggctc actgcaacct ccacctcccg 81720
    tgttcgagat tctcctgcct cagcctcctg attagctggg actacaggca tgccacgacg 81780
    cccagttaat ttttgtattt ttagtagaga cggggtttcg ccatcttggc caggctggtc 81840
    taactcctga cctcaggtga tctgactgcc tcagcctctc aaagtgttgg gattacaggc 81900
    ataagccact gcatctggcc agaaatgaaa taagtaaatc ttttaacctg ctctaacaat 81960
    atagtgaaaa gaccatatta ttattagagc aggttaaggg atttgcctat ttcgggttct 82020
    agttatagtc ttaaacttgg acattcttgt agaaagtaaa aagtttcctc ttcaaagttc 82080
    cccttcttgt taaagaatac atcataagtg ttagaagtaa tagtttattt taaagactaa 82140
    ctttcttcaa gcctccttgc tttgtgctaa taactctttg ttaagcccta tcctatgtaa 82200
    ctgttggaca tgctcacagg cacgttccag ttcacagcct atgccccttc cttatttgga 82260
    aatgttattg cttccttaaa cctttcggta agcaacttcc tctccttctt cgttcttcct 82320
    tgcacttacc tatttagaaa gttttaggct attagcaaat cggctatcag tttaagagtg 82380
    tgaggtcccg ctccagccaa tggatgcagg acatagcagt gaggacgacc caaatgcgta 82440
    agggataaat atgtttgctt ttcctttgtt caggtgtgct ctcgacatcg ttccatctgc 82500
    gattgagcac cctttctgca gaaagtaaag attgccttgc tggagatctt ttgtctccgt 82560
    gctgactttt cttcgtggca ccgattatct atttctaaca attttggtat ttctaacatt 82620
    ctgaacaatc ttgggctagt tgtctcttct gggcctgttt ccccatccgt cacatgataa 82680
    acttcattgg tttaaaaacc ccagcgaaca tttattgagt tactattacc ttcctgccct 82740
    ccccaacccc aaccccaggg agcagttaca acctcagccg ctgagcgcac tcgccgggtg 82800
    ttaagaagca ccaaagacag ggaggcttga ttgattttgc tttgggagta gagggtcaga 82860
    agattcacag gaaaatggca tttgagcaag gatgattcac tggagctagc ttttaaatac 82920
    tggcgaggct tttatgttgc agtcccttac aaagttgagc attcgcaggg actgcactcc 82980
    gaaataagcc cgcttcccct tttcattcgc taatgatcca gggagctgct ggttccgcat 83040
    gcggcaggtt gtgccttttc ctaatcaggg ttctgcatcg cctcgaaccc gcaggccgtg 83100
    gcgggttctc ctgaggaagc agggactggg gtgcagggtg aagctgctcg tgccggccag 83160
    cgcctgtgag caaaactcaa acggaggagc aggaggggtc gagctggagc gtggcagggt 83220
    tgaccctgcc ttttagaagg gcacaatttg aagggtaccc aggggccgga agccggggac 83280
    ctaaggcccg ccccgttcca gctgctggga gggctcccgc cccagggagt tagttttgca 83340
    gagactgggt ctgcagcgct ccaccggggg ccggcgacag acgccacaaa acagctgcag 83400
    gaacggtggc tcgctccagg cacccagggc ccgggaaaga ggcgcgggta gcacgcgcgg 83460
    gtcacgtggg cgatgcgggc gtgcgcccct gcacccgcgg gagggggatg gggaaaaggg 83520
    gcggggccgg cgcttgacct cccgtgaagc ctagcgcggg gaaggaccgg aactccgggc 83580
    gggcggcttg ttgataatat ggcggctgga gctgcctggg catcccgagg aggcggtggg 83640
    gcccactccc ggaagaaggg tcccttttcg cgctagtgca gcggcccctc tggacccgga 83700
    agtccgggcc ggttgctgaa tgaggggagc cgggccctcc ccgcgccagt ccccccgcac 83760
    cctccgtccc gacccgggcc ccgccatgtc cttcttccgg cggaaaggta gctgaggggg 83820
    cgccggcggg gagtcaggcc gggcctcagg ggcggcggtg gggcaggtgg gcctgcgagg 83880
    gctttcccca aggcggcagc aaggccttca gcgagcctcg acctcggcgc agatgccccc 83940
    tgagtgcctt gctctgctcc gggactcttc tgggagggag aaggtggcct tcttgcgcga 84000
    ggtcagagga gtattgtcgc gctggttcag aagcgattgc taaagcccat agaagttcct 84060
    gcctgtttgg ttaagaacag ttcttaggtg ggggttagtt tttttgtgtt tctttgagga 84120
    ccgtggatca agatcaagga aatctcttta gaaccttatt atggaagtct gaagtttcca 84180
    aatgttgagg gttttatgtc taaaagcaac acgtgaaaaa attgttttct tcacccagtg 84240
    ctgtcttcca atttcctctt tggggggagg ggtagttact gctgttacta aaataaaatt 84300
    acttattgct aaagttcccc aacaggaaga ccactacttt tgatgacttt ggcaagtttg 84360
    ctaactactg gaaccctaac ttacaaacga actacttaca tttttgattt ccagttgtat 84420
    tacctgccca atgtttacgt agaaacagct taattttgat tctgggtaac gttgttgcac 84480
    ttcattaaaa atacatatcc gaagtgagca agtatgggtc tgtggacagc agtgattttt 84540
    cctgtcaatt cctgttgctt cagataaaat gtaccagaca gaggccgggc gcggtggctc 84600
    acgcctgtaa tcccagcact ttgggaggct tggcgggtgg atcacctgag atcgggagtt 84660
    caagaccagc ctgaccaaca tggagaaacc ccgtgtctac taaaaataca aaattagcca 84720
    gggtggtggc gcatgcctgt aatgccagct acttgggagg ctgaagcagg agaatcgctt 84780
    gaacctggga ggcggaggtt gcggtgagcc gagatagcac cattgcactc cagcctgggc 84840
    aaaaagagcg aaactccgtc tcaaaaaaaa agtaccagac agaaatgggt tttgttttct 84900
    ttttttgttt tgagacggag tttcgctctt gttgcccagg ctcgagtgca atggcgcgat 84960
    ctcagtctcg gctcactgca acctctgtct cccaggttta atcgattctc ctgcctcagc 85020
    ctcccaagta gctgggatta cccatgcccc accatgcccg gctaattttt gtatttttag 85080
    tagaaacggg gcttcaccat gttaggctgg tcttgaaccc ctgacctcaa gtgggcctcc 85140
    cacctcggcc tcccaaagtg ccaggattac aggcatgagc caccgcggcc agccagaaat 85200
    gggttttgga aaaagcacta aacaaaatcg aacttggttt catatgacag ctctgctgct 85260
    aactgtaaca ggggcagacc agttaaccta cttttctgtc ttctgtcagc tgagaattag 85320
    atgattccca aaggcccatt gaactctgaa tgactttaaa tacttcttct taagtgggta 85380
    cacggttttg gtaactgatg ccaggtgatg aatgcatgaa agtgcttaat gaatgaaacc 85440
    ggtaaaatag taggaggaag ctttattggt aaggcagggg tatacctaat agctctctaa 85500
    tttattggta ttgaagtggt taacttttgt ttttttaagg ggggaaaaca ttctaagaat 85560
    aatgaggcaa actgcatatt gcacaagaga ctgttgtctc tattcaacaa ataccttttg 85620
    agtgtccaga gtctgccagg tgctgtgcta ggccctcacg attgagtagt gaaccagaga 85680
    atgtccctgc acccatggag cttattgtct actggggtag acagataata aataagcaaa 85740
    caaatcttct ctcttctccc tttcgctcca tgtaagtgtg tgtgtatagg tgtatactta 85800
    caagttgagt aaagtgttat gaaagattaa gaggagaaat gcattttggt tagatgttag 85860
    aggactcagc aggtgacctt gaaacttaga gctgaaggat cagtaggagg taactagaga 85920
    ggccagggaa tcgcatgttc aaaggccagg aggcaagaaa gagcatggtg cccttcaaga 85980
    gaggaaagaa ggctactgtg actggagcat agatgtaggc aagtgttggg tgattgagag 86040
    ctctacgggc catggttagg ttttattcct aatgccgaga tgccaaacat ggtggttcat 86100
    atctgtaatc ccagtatttt aggaggccga ggcaggaata tagcttgaac ccaggagttc 86160
    aagaccagcc tgagcaacat gagacctgta caaaacattt aaaaaattgc tgggtatgat 86220
    ggtgcacacc tgtggtccca gctactcagg aggctgaggc agaaggatca cttgagccta 86280
    ggaggtggag gctacaatga gccatatttg agtcactaca ctccagcctg gatgacaaag 86340
    tgagaccatg tgtcaaacaa aatacagaaa gaatattaat ttaaaatttt gaaagaggag 86400
    tgatctgaac ttatatctta aaaagatcat tctagggcat ggtggctcat gcctgtaatc 86460
    aagggctttg ggaggctgag acaggaggat cacctgaggc cagttcgaga tcaacctgta 86520
    cagcatagag agactccatc tctacaaaaa gaaaaaataa atagctgggt gttgtgagtt 86580
    attcaggagg ctgaagcaga aagatcactt gagcccagga gtttgaggct gcagtaagct 86640
    atgatcccac cactgcaaca cagtgagatc ttgtctcaaa aaaaaaaaaa aatcattcta 86700
    ggtgcttttt ggaggctgga tgtggtaaga gtagaagctg gagatggtcc tgttagggat 86760
    tcgattcaga ctttaaatac catcaatgca ttgagtccca aatttacatc actacgttgg 86820
    atccttgccc ctgaatccag actggtatat ccaactttag gttcagtttg tatctctacc 86880
    tgaccaatat agaggtgtcc agtcttttgg cttccctagg ccacattgga agaagaattg 86940
    tcttgagcca cacatagagt acactaacgc taacaatagc agatgagcta aaaaaaaatc 87000
    gcaaaactta taatgtttta agaaagttta cgaatttgtg ttgggcacat tcagagccat 87060
    cctgggccgc gggatggaca agcttaatcc agtagatacc ttcaacttac aatatctaaa 87120
    attttatgcc agatttagtc attttaaacc tgctcatcag tttttctcaa gaagtagtat 87180
    tttggctttt tttcttttct tttttttgag atggagtttc gctcttatcg ttcaagctgg 87240
    agtgcagtgg cggatcttgg ctcactgcaa cctccgcctc ctgggttcaa gtgattctcc 87300
    tgcctcagcc tcgcaagtag ctggaattac aggcatgcgc caccatgacc agctaatttt 87360
    tggagacagg gtttcaccat gttggtcagg ctggttttgt actcctgacc tcaggtgatc 87420
    tgcctgcctc ggcctcccaa aggctgggat tacaggcatg agccaccgct cccggctgca 87480
    tttttggatt tttagttgct cagcccaaaa ctttagtaca tctttgaacc tcttctttcc 87540
    tcctactcta tatctgatcc atcagcaaat ctgttaggtc tacctcacac atatcgaaat 87600
    cctaccacgt ctcaccatct gtgacaatta acaccctggt ctaggcagtc atctctgtta 87660
    agattgagtg gttaaggatg tcctctaagg agatgacatt caaatcttag cttaaatgtc 87720
    aagagggagc tggttttata aagattgagg aggcagcatt attttgccat aggcttccat 87780
    ttggtttcca ttccattctt gatacttatg gtatatattc aaaacaaatg cacagaaaca 87840
    gacccaggta tattgggaat ttcggatata gagttcctag ttgggaaaag atagactgat 87900
    ctgtaaatga tgctagttat ccatcatctg gcaaaaaata atttcctgcc tcctctcata 87960
    tatctcagat caacagactt tttctgttaa gggccaaatc ataaatattt taggctttcc 88020
    agaccatatg gtttctgtca cactctcctt tatccttgaa gccatagaca atatgtaaac 88080
    aaatgggcat ggctgtgcta cgataaaact ttacttacaa aaactggtag tgggccagtt 88140
    taggcatggc cagcactttg ggaggctaag gcagatggat cacttggggt caggagtttg 88200
    agaccagcct ggccaacatg gtgaaaccct gtctctacta aaaatacaaa aaatagctgg 88260
    gcatggtggt gggtgtctat aattccagct actctggagg ctaagacaca agaatcactt 88320
    gaacccagga ggcagaggtt gcagtgagct gagatagcac cactgcactc cagccagggt 88380
    gacggagtct taaagcaaaa caaaacaaaa ggtagtgggt tgtatttggc ccatgggctg 88440
    tagtttgcca atccctgatg cagaaacaaa ttccaggtaa ataagagcct ggaatgttaa 88500
    aaaaacaaaa cttgaagtca tgtagaagaa caggtagggg gaacaatcct gatctcagga 88560
    taggaaggga tattgcttaa aataagacac aggaaaatat aatccatgtt gtgtaaattt 88620
    gactacgtta aaacttaaaa ctttcgccaa gcgcggtggc tcacgcctgt aataccagta 88680
    ctttgggagg ccgaggtgag cagatcacca ggtcaggaga ttgagaccat cctggctaac 88740
    acggtgaaac cccgtctcta ctaaaaatac aaaacattag ccgggcgtgg tggcgggcgc 88800
    ctgtagtccc agctacttgg gaggctgagg caggagaatg gcctgaaccc gggaggcgaa 88860
    gcttgcagtg agctgagatc gcgccactgc actccagcct gggcgacaga gtgagattcc 88920
    gtctcaaaaa aacaaaacaa aacaaagcaa aaaacctaaa actttcatac aataaagtat 88980
    acctaagata cttctagaag agaagattta catccaggac gtgtatggaa tttctgcaag 89040
    taataagtaa aagacaaggg acatgaagag gcagttcaca aaagaggaag ccaaaatgac 89100
    caataaacat gaaaggatgt ttaacctcaa aggaaacaag gaaatgaatt aaaaacatca 89160
    aatgccattt caaaactagt aagttggcaa aattaaaaat accaaggatg agaatatgaa 89220
    gcatggctat atgagtgcat ggaatggtac agtcactttc attaaaaatg cacataattt 89280
    gttttttatt tatttttttg agacagtcta tgtcgcccag gctagaatgc agtggcatga 89340
    tctcggctca ccacaatctc tgcctcctgg gttcaagcaa ttctcctgcc tcagcctcct 89400
    gagtagctgg gattacaggc acatgccaca acgcccggtt aagttttgta tttttagtag 89460
    agacagggtt ttgccatgtt ggccaggctg gtctcgaact cctgacctca ggtgagctgc 89520
    ttcccaaagt gctgggatta gaggcgtgag ccaatgctcc tggctgaaaa aaatgcacat 89580
    aatttgttac ctagcaattc catgtctaga ggcttatcct agagaaattc ttgcttatat 89640
    gcataggaag acgtgtacta gaatgttcac tagttgaatg tttaagtgaa aattaggaaa 89700
    taaagtaaat gttcattaac aggaaaatga gtaaaggtat atttataaaa caattaagta 89760
    gctaaaatga ataaactaga gctgcgtgaa tgaactagaa ctggttcaat agtcatgtca 89820
    gattattgaa tgaatacagg tcagatatgt atagagtgtc atttgtgtaa ttaatttttt 89880
    tttttttttt gagatggagt ctcactctgt tgcccaggct ggagtgcagt ggcgtgatct 89940
    cagctcactg caacctccac ctcctgggtt aaagtgattc tcctgcctca gcctcccgag 90000
    tagttgggat tacaggcatg caccaccatg cccagctcat tttcctattt ttagtggcca 90060
    cagggtttca ccatgttggc caggctggtc ttgaactcct gacctcaagt gttccaccca 90120
    acttggcctc ccaaagtgct aggattacag gcgtgagcca ccgtgctcag ccatttgcgt 90180
    gatttttaaa gatgtgcaga ataatgccat taaaaaaaat acacatacat gtatatatat 90240
    acacgtttgg ctgggtgtgg tggctcacac ctgtaatccc agcactttgg gaggctgagg 90300
    caggaggatc acttgagccc aggtgtacaa gactagcctg ggcgagatag caagacccca 90360
    tctcaacaac agaaaggata attaggtatg gtggcatgag aggatcactt gagcccagga 90420
    gttcgagtgt tatcaggcca ctgcactcta gcctggacaa caaagcaaga ccgtgtctca 90480
    aaaaaataaa aataaaaagt atttgtatgt ggtcatagtc aaaaaacgta catggaagga 90540
    aaatgtcttt atttatttat ttattttttt ttttttaaga cagagtcttg ctctgtcacc 90600
    caggctgggg tacagtggtg taatctcagc tcaccgcaat ctcggcctcc cgggttcaag 90660
    cgattcttct gcctcagcct tctaagtagc tgggactaca ggtacccgcc accacaccct 90720
    gctaattctt gtgttttcag tagagacagg gtttcaccat gttggcaagg ctggtctcga 90780
    actcctgacc ttaagtgagc cacccgcctt ggcctcccaa agtcctggga ttacaggtgt 90840
    gagccactgc gcttggccag gaaatatcta atttagtaag tatttatatc tgggaaagga 90900
    agggtcaggt ggtgattcat aggaactcta aagtctatgt ataatactta gggggacaga 90960
    aggaaataaa gcaaaatgct gatatttgat tgttgagttg tgtatatgtt agaagtataa 91020
    cataggagat ctgattgata gtaggagaat gtttttaggt ggtaaaagtg gaaccgtggt 91080
    ggtttgtttt ggcagtagaa tcagttggtc atagtttgta tgtggaaggt aataaacaga 91140
    ccatgttaag gatgacttcc ggaattttgg tctgagtagt gggtggatga cagtgtcatt 91200
    catgagggaa gatgaagact gaggtaggaa caggtttggg agaagatgac atgttccctt 91260
    ttagacaagt ggaattatgg aagatggcag gtaggtggtt agctatatga atttgagata 91320
    aaagatttag gatggagata taaatttagg agtaacagcg tatctatggt attgtaagcc 91380
    ttaagaatgg gtaggatcag ccaggaaata cagatgtata tgcagaagag aggagtcaag 91440
    gaagccaaga caagttaatg tttaaagtga gtgatgtagt ccatgggcag atgctgctga 91500
    gagggctgca aacaccagtg accctacaac atttttaaat gtcgtcttcc tgacagcagt 91560
    gatcagtacc tgcaacgatc ttatttattt ttttcatgtt agtctccaca cacttgaatg 91620
    tagacttttt gaaggcaaaa tcattgcctt ttctgagctg ggagcatgtc tggcacatac 91680
    caagcactca acagttgatg tattgacttc atccagatac tctgagggcg agttatttcc 91740
    tgctactagc ctttcacctt tcaatgttta agagcacaaa tacagagatg ggcacgtttt 91800
    ggcatttctt attttgataa ccttttcctg gtaagatttt ttaatgttga aaaaaaaaaa 91860
    caagaaaaga gggttaaaaa tagtcttatg tcagatcctg tgatagaatt cacacttggc 91920
    ttaagctgct gggcaccttc ctatcttgga tgtcatatta gcttatctac agcagaattt 91980
    ttactgtttt atgtagtaag gaagcaatta tatgattatt ttacagacaa attattcttt 92040
    atcttttatt tttttagacg gagtctctct ttgtctccca ggctggagta cagtgtcgcg 92100
    atctcggctc actgcaacct ccgcctcctg ggttcaagca attctctgcc tcagcctccc 92160
    aagtagctgg gcttacaggt gtccgccacc acacccagct cattgttttg tatttttagt 92220
    agagatgggg tttcaccatg ttggccaggc tggtcttgag ctactgacct caggtgatcc 92280
    acccgccttg gcatcccaaa gtgctggaat tacaggcgtg agccaccgtg cctggcccag 92340
    acaaattatt atactctgag tgttagaggc ttaggatgtt ttcacttgat gctatgggag 92400
    gaataagtaa taagatatga tacacaacca aagacctttc ttcactatgc ttctagtagc 92460
    tagtactatg gatgacacat ggtaataata ttggttagca tttgtcctca atttactgtg 92520
    ctagttactc ttctaagccc cttacaggta tatatttttt ttcatcaata atcctctaag 92580
    gtagttttta ttattgacct aattttataa atcaagaaaa ttaagaccca gagaagtaag 92640
    taacttgtcc aagatcacat ggcttataag tggtagagcc agaatttgac cccagatgtt 92700
    gtgactacat tgtctctcca taagcaggtt caactctttt gactggatgc tgttccaagg 92760
    tcacttcctt agagaagcct ttgctgacaa ctaccctcct gtgccctcct ccaaggctgt 92820
    ccattgttct agaactttga atactcatct tagaataaag ctggtctaat ttttacagtg 92880
    ttatagaatg gatctctgac tgcaaaagtt ggtcataatt atctttttat gttctagtga 92940
    aaggcaaaga acaagagaag acctcagatg tgaagtccat taaaggtaag ttctgccctt 93000
    ggcagtccac tgcattaaaa agtgatgtgc tttgcatttg tgagttcttt aatcctgtta 93060
    tactctctct tttggcatta atcatttctg ccttatttta taattactta tgattttgat 93120
    ttatttccct ctttaacctg tataatgctt taacatctag catataataa gtaggctttt 93180
    tttttttttt tttttttgga gacggagtct tgctctgtta cccaggctgg agtgcagtgg 93240
    cgcgatcttg gctcactgca agctctgtct cccgggttca caccattctc ctgcctcagc 93300
    ctccccagca gctgggacta caggtgcacg gcgccacgcc tggctaattt tttgtatttt 93360
    ttagtagaga cagagtttca ccatgttagc cagtatggtc tcgatctcct gaccttgtga 93420
    tccgcccgcc tcggcctccc aaagtgctgg gattacaagc gtgagccacc gcacccggcc 93480
    gtaagtaggc tttttttacc ttaattttat ttttttgaga tggagtcttg ctcttatccc 93540
    caggctggag tgcagtggtg ccatctcggc tcactgcagc atccacctcc cgggttcaag 93600
    cgattctcct gcctcagcct cccgagtagc tgggattaca ggtggccgcc accatgccca 93660
    gctaattttt gtatttttag tagagacagg gtttcaccgt gttggccagg ccagtctcaa 93720
    actcctgacc tcaagtgatc cactcgcctt ggcctcccaa agtcctggga ttacaggcgt 93780
    gagccaccat gcctggccat aagtaggctt ttactgagcc ttgtgtgtat tggctatcct 93840
    agtgattaca gtgaaccagt gcccttctta ttaatcacac atttaattgt tccctaaaag 93900
    tgattagttc actttattta tttagtaaga caaaaaatga agaatactct taactgagca 93960
    gtctgttaac tgtaggaaag cactgacact tataaggctt agttttctgt catttatcca 94020
    gaagtatggt tgattacagt ttttactttt ttatttgaat gaacaacctt aatttaaaat 94080
    atattttgtt tattttttgt tgggatcgat acattgtcct tgtttataga ttagagcatg 94140
    ctttttaaag atgctgtatt actcactgat tttatttgtc cagtgtacag agattgaagt 94200
    gggaaaatta taatggaaat tgtttccata gtcattacat attaatttca tcaatttatt 94260
    tccataaaat ctgtagattg ctacttattt agatttttcc ttcaaatgtt tttatgttgt 94320
    attgcttgca ctgagtattt attctatatg ctcaatttgc tggagaagaa gactaattat 94380
    aacttaggca agttgtaaaa ttagggaaaa aagtaaggta ccttacagcc tagtttactt 94440
    atttcttatg taaagccagt tagattccac attagttcaa actgccttct ttgagcaaaa 94500
    cttgattggc agtgataaag gcttaaagcc cttctcaagc agagacctgt aaagactaga 94560
    tctgactgta gtagaaggaa ggaacttaga tgtttcaggc agtgagaaca ccagtcttcc 94620
    actctaaact ttgccactaa cagtatgacc ttgggaagtt gtaactttct tcagattctt 94680
    catttgttga atggggggat tggcctagct aatttctaaa tctctactgg gctaaaaaat 94740
    tctgtgctta tactctgatt atgaagtaca taatctgtgc ttaacattca ctgacttatc 94800
    cttaggataa tacagaagca gtacaagaaa cagcccctca agatgtttgc agtctggtta 94860
    gaaagacaaa cttatacaca gaacagtagc aaatagacca aaataataat agctgccatt 94920
    tatagaacac ttcttctgtt ctgggcatta gacaaaaact gactataacg gtgaacaaaa 94980
    aagacttagg tcctgccctc attgaactta cagattagta ggggagagga acattaatca 95040
    agtaattcca cagatggctt agcctagatt ggtagtgatg gaagtaaaga gatgtgaacg 95100
    gacttgaaaa aaaattcgga ggcaaaatgg atagaagttt attattgatt aaatatgagg 95160
    tgtgagagag agggatattt aagattgata cctaccttct ggcttgccta acagaaccaa 95220
    aacaggaaat tatatgttca gttttgttat gttgggtggg aggtgctttt gagtcattca 95280
    tttatatatg ttatatatgt tattttatat gcatagtaat tttaaggtct gagttttaaa 95340
    ccaaaggtta gagagtgatt ttttagagtc tagcaaacct aagttgaaat cctgcctgtt 95400
    gaaatggctg tttactagct cattaaccta gggcaaagta ttcaacttgt tttcattttt 95460
    gtcttcatct ctaaaatgag gaaaatatgg tcttacaaga ttgtcctgag agatagatga 95520
    aataatatcc aaaaaaaaaa aaggtacata gagaaactcg tatagtgcct ggtatatagt 95580
    aggtcctcca ttggtagcta tcattatcta gttttaacat agccttcagt ttgttgaatt 95640
    agtcaaactg agtgaagcac tgcaaggaat tcagaggaat ttgagatcaa caaatgattt 95700
    ctgaagttta gggaagactt catggcaatg acacttacct tgtataaaag ttgaagaata 95760
    agaaagattt gaatgagaga ttctttctct tctccctacc agcccagctt cttatttgag 95820
    gatatattgg gcaaaggggc cttcagacaa gtagagggag atttttacag aaagattgag 95880
    atgaaggtat agaaggctgt aaagaccaga aaagagaatt gagacagagg aagcaggaag 95940
    ccactgtagg tttttgagca agatattgat gctgtaagta tggtgtttat gaaaggttag 96000
    tctggaagag atttgcagga tggagacccc ggaagttttt ttgttataat acagaaagac 96060
    ttgcactgag ggtgaggtgt taaaaataaa caggtaagta aatgtttaaa catcttgaag 96120
    gaaaagtcaa caaatcttgg caagtaaaca gataacagtg aaaaagaatg ggaccaagat 96180
    tttgagtttt ggagactggt ggattgaaca gacagggaaa ttgagaggag aatcagatga 96240
    tgatgtttta agttgatatt tagacagatt gtgcttgaga tggtaaagtc aatgtgggtg 96300
    ggaatgctta gtagcgagta atcagtgata caagaccaaa gcccaggtca aagacaagtc 96360
    acagatacag atcagggctt tttcatctgc tccacagagg tgtaccctag gagctgttgc 96420
    aaacagtcca tgtggagggt gtgagtaaga tgtttccctt gaatttgcca gaattacttt 96480
    tttgttgttg ttgttgtttt ttctgagaca gattctcgct ctgttgccca ggctggaggg 96540
    cagtggcgag atcgcgcagc tcactgcaac ctctgcctct cgggttcgag tgattctcct 96600
    gcctcagcct cccaagtagc tgggattaca ggcttgtgcc accaagccca gctaatttct 96660
    tttgtatttt tagtagagat ggggtttcac catgttggcc agactggtct cgaactcctg 96720
    gcctcgtgat ctgcctgcct cagcctccaa aagttctggg attacaggcg tgaaccactg 96780
    cacccggtcc cttgttaagt ttattttggt gggaagcaaa ggaggtttca gcttttaaaa 96840
    agtttgaaaa ttattgctct ggtaataatt aaagatttga gagtaaatat gctttctagc 96900
    agaaagaata aaagaagaac agatagcctc aagaagggga gccaaagaag caggctatat 96960
    ctgacacact gggtgttgat aaatgggtat taaaagaatg agagcaatga gcagatagaa 97020
    gaggaaatta ggagagtata ataccatgga gaccaagaaa gatagactat caggaaggag 97080
    tggtaaaaat aagttactag ttctaagaga gatgttaaga gggaccgggg aaagccttgt 97140
    acaaatgagt tagtagcatt ttacattata tacatctaat taagaaacaa tgcgagagtc 97200
    tcaccattcc tatagactct tacttgtact tgtctgaaca cgaaaactgg cttttgttta 97260
    taaataagct aaaaattatt ttgctccaat ttctcatgaa aataaaaata aaccttcttt 97320
    taacattgaa aaaatagttt gaagacagtc actcttcatt ttgtaattcc cacaactatt 97380
    attgaatgac tgaaattatc tttattctga agccaaaggg gtgatactga tatttcttca 97440
    gactactaaa aatatatttt atgaattttt agtgtgcttt atcttttttt gttttttttt 97500
    ttgagatgga gtttcactcc cgttgctcag gctggagggc agtggtgcaa tctcagctca 97560
    ctgcaacctt cgcctcccag attcaagcaa ttctcctgcc tcggtctccc aagtagctgg 97620
    gattacaggc acctgccccc acacccagct aattttttgt atttttagta gagacagggt 97680
    ttcaccatgt tggtcaggct ggtcttgaac tcctgacctc aggtgatcca cccaccttgg 97740
    cctcccaaag tactgcgatt gcaggcatga gccaccatgc ctggcctgag gaatattttt 97800
    ctaggttccc cccaccccaa gcatttattc tgcaatttta gttttgttcc taaagcaagc 97860
    aaggtttaag gatttaaaaa taatccgtat tttagaatgc tttctggctt tgttactttt 97920
    tatccacagt agaagttctc agagaatgat ctccctcttt taatttaact ttttggcaca 97980
    gtattttgag aattataaat aatattagaa tgttttctgg ctgggtgtgg tggctcatgc 98040
    ctgtaatcct ggctacttgg gaggctgagg caggagaatc acttgaacat gggaggcaga 98100
    ggttgcagtg agccgaggtc atgccactgc actccagcct gggtgacaga gcaagactct 98160
    gtctgggaaa aaaaaaaaaa aaaaaaagag tgttttcttt cctattttcc accacttgat 98220
    taagttactt ttcctcttaa gtattttttg ctgagtatgc tgacttaaga gtaatgttac 98280
    aaaatttaat ttttaaagtt ctctgaaagc ccctttatga gagttttagg ctatcaaatt 98340
    gtgtttaatt cttaacaatt ttttgaaaaa ttatagcttc aatatccgta cattccccac 98400
    aaaaaagcac taaaaatcat gccttgctgg aggctgcagg accaagtcat gttgcaatca 98460
    atgccatttc tgccaacatg gactcctttt caagtagcag gacagccaca cttaagaagc 98520
    agccaagcca catggaggcc gctcattttg gtgacctggg taagtaacta tcatttttta 98580
    ttaacttgta ttagaaggat ttgagtacaa tatgtgaaac ttctgtcata ggatacagaa 98640
    ctatataatt ggaaagtgct ttggaaaaaa tgtatttaaa ataacagcta caagtataat 98700
    gggtagctgt gttgtgttcc tgtaaatata gaatataaag catgcccagt agaaaaacaa 98760
    gcatttccag aagaaatata tctgatcact aaatataaat atatgaaaaa gatgtctcac 98820
    tttattactg agggaagtgc aaattaaaat aatcagttaa tgttctccta acacattagc 98880
    atatttttta aagtttgaca atttgaatgt cagtgaagat gcagggaaat acccctccta 98940
    tttagtgata atataatctg gtgaagactc tttggaaagc aatttggaaa tcagtataaa 99000
    atatgcatgt catttaggcc actctttcta agacctagcc ctcagatatg ctcattcata 99060
    tgtgcaggtg tgtatgtgtg tgtgtgtgtg tgtgtgtgtg tgtatatgta tgtatgtatg 99120
    tatgtatgta tgtatgttga aggctattca ttatagtatt gtttgtgata gcaaaaaatt 99180
    atggacaaca tataaatatc tgttataggg aaataaccaa attgtggtat acgcatgctc 99240
    tggagtataa tatagccatt tgtttctatt tatttatttt cttgagacag ggttttactc 99300
    tgttgcccag gctggagtgc agtggtatga tcatggttca ctgcagcctt cacctcctgg 99360
    gcacaagcca ttctctcgcc tcagcctcca gagttactag gactgcaggc atgtgtcacc 99420
    acacccagat aattttttaa ttttttgtag agacagggtc tcactatgtt gcctaagctg 99480
    gtctcaaact cctggcctca agcaattctc ccacacaggc ctcccaaagt gctgggatta 99540
    ccaacgtgaa ccaccacacc tggttcagtg tagccattta gaaatctaaa aaagacgtgg 99600
    gaaaatgtct aaggcatgtt taaatgtgag aaaagcaagt cacagtatgc atggtaaaat 99660
    ccgttatatt aaaataagtt cttccaaaac aaaaacatat gcaggagacc tttattttgt 99720
    cagtatttct tacccaaatt tctgcactta gaaaattgca tgtcatgttg tcataagttg 99780
    aaaaaaagat ccatgaacca atggacttct aataaaatca gtcctgcttt tgacatctct 99840
    ctctactttt gtgtatattc aaaccagagt gtcaatgtgt ttgtggggca cacttagcaa 99900
    taatacatag cagacaaaat gcatatagct cagagagtaa aattgtaagt tttgctagat 99960
    cactcataaa ttgctgatga gaatttaaaa tggtgcagat gctctggaaa acaggcagtt 100020
    tctttctttc tttttttttt tctttttgag acagggtctc actctgttgc gcaggctgga 100080
    gtacagtggc gtgattacaa ctcactgcag cctcaccctc ctcaggttca ggtgatcctc 100140
    cctcagtctc ctgagtagct gggactatag gcatgcacca ccacgcctgg ctaatttttg 100200
    tatttttttt tttttttttt gtagagacgg ggtttcgcca tgtttcccag gctggtctca 100260
    aactcctgga atcaagcgat ccacttgcgt aggcctccca aagtgctggg attacgggcg 100320
    tgagctactg tgcctggcct aggcagtttg tttgtttgtt tgtttgtttg tttatttatt 100380
    tgtagacgga gtctcacagg ctggagtgca gtggcccaat ttttggctca ctgcaacctc 100440
    cgcctcccag gttcaagcta ttctcctgcc tcagcctcct gagtagctgg gatgacaggt 100500
    gcctgccata atgcctggct gatttttgta tatttagtag atatggggtt tcaccatgtt 100560
    ggtcaggctg gttttgaact cctgacctca ggtgatcagc ccgcctcggc ctcccaaagt 100620
    gctgggatta caggcatgag ccgtcatccc tggctggtgg tttcttatga cgtgaaacat 100680
    gcaattacca tatgacctag cagttgcact ctgtatttat cccagataaa tgaaaactta 100740
    ccttccaata aaaacctgtg cacaaatgtt catagcagct taatattgaa aaactggatg 100800
    ttcttcagca ggtgaatgaa ctggttcatt cataccatgg aataccattc agcaataaaa 100860
    aggaacaaac tgttgataca tttaaccacc tggatgaata tcaagggaat tatgctgtca 100920
    gacaaaaacc agtccctaaa gactacatat agtatgattc cgtttggata atattcttga 100980
    aatagagaaa ttaagagaaa tgaaaagatt agtgtttgcc agatgttaga gacagggagg 101040
    tgagaggggt aagtgggtgt agttataaaa gtgcaacatg agggatcttt gtgatgttga 101100
    agttgtatct tggcagtgga tgcagaaatc tcaatgtgat aaaattacaa agaactaaaa 101160
    acaagaatga gtatagataa aactggggaa atctgaacaa gttagagtgt tgtatcactg 101220
    tcagtatctt agagtgatat tgtactatag ctttgcaaga tgttaccatg ggagaaacta 101280
    aagtgtacaa gggatctcta ggtattatta tttttttaga gatggggttt cactatgttc 101340
    cccaggccgg tcttgaactc ctgggctcta gtgatccgcc tgccccagcc tcctaaagta 101400
    ctggaattac aggcgtgagc gaccatgcct ggccctttca gtattgtatc ttagaacttc 101460
    atgtgaatct agcattatct catagaattt aattaaaaga aattgtaaac ctcacagaag 101520
    atcagaattt cctcaagttt gtgatgttga caaagatgaa ctagttgaca ctgacagtaa 101580
    gactgaggat gaagacacga cgtgcttcaa aaaaatgatt tgaatatcaa tggattaaga 101640
    agaactcttt tgacaaattg atgaaaccct cagtcagttt tataagaatg cccatcttta 101700
    tgatcatgct atgaaagcca atttttaaaa aaattttttg tctttcctaa caattagctt 101760
    gtggttataa tttaaattta gttaaatata agataaatga ttttttatta agtttagttt 101820
    catttttcaa ggtacgatct caaagctact ctttaaccta ctatgaatga ataatgctga 101880
    gttcataaca tctttgtaga tatatccaca attttccctc aggataagtg cctacaagtg 101940
    gaattactgg actgaaaata atgcagtttg ctaagacttt gctatctgtt cctgaatgct 102000
    cctccaaaaa ggttttgcca gtttacatcc tcatgaccag cgaatgagag tgttgcctat 102060
    tttcctgtgc ccttgttact gcttaataat ttttgaaaaa aatctaattt gacagacaaa 102120
    aatgcatttt atgttaattt gcttttctgg gatttttaat gaggttgagt atagttttta 102180
    atatttttat tggccccttt ggaactagta tcataagttt tttttcttaa gaatttatgt 102240
    agtctgggct gggcgcagtg gctcacgcct gcaatcccag cactttggga ggccgaggtg 102300
    ggtggattgc cgaaggtcag gagtttgaga ccatcctgac caacatggtg aaaccgaatc 102360
    tctactaaaa gtacaaaaac tagctcagcg tggtggcggg tgcctgtaat cccagctact 102420
    taggaggctg agtcaagaga atcgcttgaa cccgggaggt ggaggttggt tgcattgagc 102480
    cgagatcgcg ccattgctct ccagcctagg caacaagagt gaaaagtctc aaaaaaaaaa 102540
    aaaaaaaaaa aaaaaagaat ttacatggtc tgaattgcca ttaaaagaga tatgagaatt 102600
    attgagtaac aaataacttt ttaataattt aggcaagttt tggacgattg tactttgttt 102660
    agaaaccaaa agcatagtat ttgtagtttt tttatttact ttagttgcta ggaagtaaac 102720
    tttattcaag gtctctggta ccagttgttg ctaaaagtga ttgactaatc tgtcaatctg 102780
    aaattatttg ttgctgaact gctaattctt ttgcttctat cttttaggca gatcttgtct 102840
    ggactaccag actcaagaga ccaaatcaag cctttctaag acccttgaac aagtcttgca 102900
    cgacactatt gtcctccctt acttcattca attcatggaa cttcggcgaa tggagcattt 102960
    ggtgaaattt tggttagagg ctgaaagttt tcattcaaca acttggtcgc gaataagagc 103020
    acacagtcta aacacagtga agcagagctc actggctgag cctgtctctc catctaaaaa 103080
    gcatgaaact acagcgtctt ttttaactga ttctcttgat aagagattgg aggattctgg 103140
    ctcagcacag ttgtttatga ctcattcaga aggaattgac ctgaataata gaactaacag 103200
    cactcagaat cacttgctgc tttcccagga atgtgacagt gcccattctc tccgtcttga 103260
    aatggccaga gcaggaactc accaagtttc catggaaacc caagaatctt cctctacact 103320
    tacagtagcc agtagaaata gtcccgcttc tccactaaaa gaattgtcag gaaaactaat 103380
    gaaaagtgag tatgtgattt tcttgtgtgt acatatgtgt ctcactttct ttttttaatt 103440
    tactaagcag aacttcagat gaggaataaa atgattggaa tatttttttt ctcctctaac 103500
    tacttgtaaa tttgggagaa tttggagagt gtagtagagt cagatcagtg tatggaaaag 103560
    gagcaggagt gactggacct tctaagaagt gtgttatcag aattagtaaa tgaagggtca 103620
    aatgtcctac ttttcccctc cactgatttt gacatcaaac cattatccac atagccttat 103680
    ttcctccctc ggtcttaatt ttattaatat tttactgcac tttgcagata aaatttttaa 103740
    aaaattttta aaaattgcca ataagtgaca tttattaagt tcagtgctta gtgtatattt 103800
    ggattttatt tattagtcac aagacctttg tgcaggtagt aggcatgatt atcttttttt 103860
    ttttgagatg gagtcttgct ctgtcgccca ggctggagtg caatggcgcg gtctcggctc 103920
    actgcaacct ccgggttcat gccattctcc tgcctcagcc tcccaaatag ctgggactac 103980
    aggcgcctgc caccacaccc ggctaatttt tttgtatttt tagtagagac ggggtttcac 104040
    catgttcgcc aggatggtct cgatctcctg actttgtgat ccgcctgcct cggcctccca 104100
    aagtgctggg attacaggca tgagccaccg cgcccggact gattatctta tttacacatg 104160
    agaaaaccag ggcttagaaa ggttaggtaa cttcctctag gttgtacagt aaatgtggac 104220
    ctagaagcat tttgacaaga gcacctgttt ttttttcttc tctattagtt tagaaattat 104280
    atactcttaa ttatcacctg ggattttgat tagacagcct tcatgttctt tttcatctta 104340
    aatgttcttt gtgtcttaaa gggctaagtg atttcttcag atcttttagt tcactcattc 104400
    tcagtgaact aaaatgaggt ctaatctgct actgaatcaa gttttcagca tgttatttcc 104460
    ttcctccctc cctccctcct tccttccctc aaccaggctc ccgaggagct gggattacag 104520
    gcgcccgcca ccactcctgg ctaattttta tattttagta gagacggggt ttcaccatgt 104580
    tggtcaggct gatcttgaac tcctgacctc aagtgaccca cctgcctcgg cctcccaaag 104640
    tgctgggatt acaggcatga atcaccacac ctgacggcat gttattttca tcgcaaagtt 104700
    actgtaagct gggagaagtg gcacacactt gtactcccag ctactcagga agcttaaggt 104760
    gagaagattg cttgagccca ggagttttga gaccaacctg ggcaacacag caagacccca 104820
    gctcaaacaa agaaaaaaag ttattgaatt ttttatttct atggatcatt ttttgtagtt 104880
    tcttattcct ttcacccttc attcccactt ttgatcccat cttttattta tttagtttta 104940
    ttaaatgtat atttgtctga taattctgct atctacagtt ttttgtggac ctgactcagc 105000
    atttctttgt ttcttcggat tcagactgtt ggtggcttgt gattttagtg atttttggcc 105060
    gtgaacatgt ttcttggact tttgtctgtg ggaattctct gtgtactctg tataaattaa 105120
    gttacttcag gtgttttgca ttttcttttg ccatgcacct ggggcctggg tcactaccct 105180
    tctggtacca cttaaaactg aatttttgtc ttgggtgctc gtactgatcc tgtatgagta 105240
    caggtttata cttactgtag aaatatggtg tttgattatg gggtattgtc ccagatggtg 105300
    ctggagtatt aatatgctct ctgttaaact taatgtgttg tccctgtaaa actccaaaat 105360
    tctgaattcc agaatactac tggccccaaa tgtttaagat aagggcactg cctgtatttg 105420
    tttctgcctc ccactatttt ccttagttta acacaaactc acctttttaa aaaacatttt 105480
    gagagaattc agtattggga agagtttcta acctgtttct ggaaatggaa gtccaaagtc 105540
    tgtttctgta attgtttttt ttttgagatg gagtctcact ctgtcaccca ggctggagtg 105600
    caatgacgta ctctcagctc actgcaacct ccacctcccg ggttcaagcg attctcttgc 105660
    ctcagccccc tgagtagctg ggattacagg tgcccaccac catgcctggc tgatttttgt 105720
    atttttagaa gagatggggt ttcgccatgt tggccaggct ggtcttgaac tcctgacttt 105780
    gtgatctgcc cacctcagcc tcccaaagtg ctaggattat gtttctgtaa ttgtaataca 105840
    tttattgttt ttagaaactg tctttgcttt agtggtaatt ttcaataaaa atagaaatag 105900
    cagtggagtt attaaaagag cattagttac atttttccct ttttcattat cttcaaatat 105960
    tatatatagt aagtttgacc tttttaaaat gtatacttgt atcagtttta acacatacat 106020
    agattcctgt aactgtcacc actataaggg taaagaacag ttagttcctt cacctttgaa 106080
    gtcaagcccc acctctatcc caacacttgg caaccgctga tctttctccg tctcaatagc 106140
    tttgcctttt ctcttttttt ttcttatttt tttttttgag acagcgtctt gctctgtcgc 106200
    ccgagctgga gtgcagtgag gcaatctcgg ctcactgcaa cctccgcctc ctgggttcaa 106260
    gcagttctcc tgccttagcc tccctagtag ctgggattat aggcacgcac caccacaccc 106320
    ggctgatttt tttgtatttt tagtagaaat ggggtttcac catgttggcc aggctggtct 106380
    caaactcttg acctcaagtg atccacctgc ctcggcctcc caaagtgctg ggattacagg 106440
    cgtgagccac tgtgcccaat caggactttt tttttttaaa tttacattca acttgtcatt 106500
    tttttcttgt atggattgtg ccttcagagt cacacctaag agccctttgc ctaagcaaag 106560
    gtcatgaaga ttttctcata tgtttccttt taaaagtatt gtggttggcc aggtgccatg 106620
    gcttatgcct gtaatctcag cactttgaga agctgaggtg ggcagattac gaggtcagga 106680
    gatcgagacc atcctggcta atgcggtgaa accccatctc tactaaaaat acaaaaaaaa 106740
    aaaaaaatta gccgggcgtg gtggcgggca cctgtagtcc cagctacttg agaggttgag 106800
    gcaggagaat agtgtgaacc cgggaggtgg agcttgcagt gagccgagat cgcgccactg 106860
    cactccagcc tgggcaacac agtgagactc catctcaaaa aaaaaaaaaa agtattatgg 106920
    ttttacactt tacgtttaga tatatatctt ttttgagtta atgtcgtata agtatgaggg 106980
    ttacgtcaga ttttttgttt tttgtttatt tttacatatg gatgtctagt tgttctaata 107040
    ccatttgttg aaaagacaac ctttactcca ttgaattgcc tttgtacttt tgccatattt 107100
    gtctaggcct gtttttggac tcctttttct gtttcatgat gtgtgtgtct attcctttgt 107160
    taataccaca tggtcttaat tactgtatag taagtcttaa aattgggtaa tgctggcctt 107220
    ataaaacgaa ttgggaagtt tttattttta ctcttatttc cattttctag aagagattgt 107280
    gtagaattgg tgtcatttct tctttagata tttggttgaa ttgggaagtg atgccatctg 107340
    ggcctagggt tttgtttttt gtgtgtgaga cagagtctca cttctgtcac ccaggttgga 107400
    gtgcagtggt gagatcttgg cttactgcaa cctctgcctc ccaggttcaa gttatcctcc 107460
    tgcctcagcc tcccaaatag ctgggattac aagcgtgtgc caccatgccc gactaatttt 107520
    tgtattttta atgcagacag ggtttcacca tgttagccaa gctggtctcg aacttgtgac 107580
    ctcaagtgat tagcccacct tggcctccca aagtgttagg attatagatg tgagccaccg 107640
    tgcctggcag gggcctaggg ttttcttttt cagagtattt taaactatga attcagatta 107700
    tttaatagat ataggactat ttaagttatc tgtttcttct tgagtgaatt tttactgtag 107760
    tttatggcct ttgagtaatt aattgtattg aattgtcaaa tttatgagcg tgtaattatt 107820
    tatagcattt cgggtttgta gtggtatccc tcttttattc ctggtgttgg caattgtgtc 107880
    ttgtttttct ttgtcagatt gtatagggat ttattagtct tttcaaagaa ctagcttttg 107940
    ttttgatttt tctgttgttt tgttttcaat tttattgatt ttctgctctt tattatttct 108000
    tttctattat ttctgcttgc tttgggttta ttttactctt ttttttttct ccaagttgct 108060
    taaagtagaa acttagattt ctggtttgag acctttcttt tctaagataa gcatttaata 108120
    ctgtaaattt ccttctaacc actgctttag ttacaccccc acaaattctg gtattttgaa 108180
    ctgagcacaa atgaaatgtt ctaatttccc ttgaatctta ttcttttacc aatgaattat 108240
    ttagaaatat gttatttagt ttgcaagcaa ttggagactt ttttcctgtt atttttctac 108300
    catttatttc tcatttcatt atattatggt cagagaatat attttgaatg atttcattta 108360
    ttaattttta aaaataacat taaaaaattt tttaaaatgt gaatatacca catacagtat 108420
    aaagattgta cattctgttt ttggacagtt ttctataaat gtcaagttga tttagttggt 108480
    taatgatggt gttcagtttt tctttattct tgctgatact ttgtatgcag ttatatcact 108540
    ttattactca gaagagtgtt gaactttcca actacaattt ttttttccaa ttttactttc 108600
    agctctatct ggttttgctt catgtatttt gaggctctgt tgttaggtgt gtacacattc 108660
    aggatgatat cttctgggtg aattgcctgt tttatcatta tgtaattccc tctttatggt 108720
    aattttcctt gttctaagat cagaaatatc tgttgtccaa tttatataga cactgcagct 108780
    ttcatttgat tagtgcttgc atggcatatc tttttccatt tttttacttt tgatctacct 108840
    ttataattct atttaaaggg ggcttcttgt aggcagcata tagttgggta gtgttattta 108900
    tttatttatt tatttattta tttatttatt tattgagaca gagttttgct cttgttgccc 108960
    aagctggagt gcagtggtgc aatcctggct taccacaacc tccacctcct gggttgcagt 109020
    gattctcctg cctcagcctc ccaagtagct gggattacag gcacgcgcac catgcctggc 109080
    tgattttttg tatttttagt agaaacggat tttcaccatg ttagccaggc tcgtcttgaa 109140
    ctcctgacct caggtgatcc acctgctttg gcctcccaaa gtgctgggat tacaggcgtg 109200
    agccactgca cccggctgag tcatgttatt tttaatcttt tctcacaata cagggttttt 109260
    gttggtaaat ttaattattt taatataaat tttagtataa ttatttacat taaatgtaac 109320
    tgttgcactg gggtatttat aatgtgtaaa tataattatt ggtattaata taattatatt 109380
    actcataata atattaatat ctttggattt agattaccag tttagtatat gtttttctgt 109440
    ttctccctct ttgatttccc cttttttgct tttttttttt ttttaattct tatttttttt 109500
    tagtatttgt tgatcattct tgggtgtttc ttggagaggg ggatttggca gggtcatagg 109560
    acaatagttg agggaaggtc agcagataaa catgtgaaca aggtctctgg ttttcctaga 109620
    cagaggaccc tgcggccttc tgcagtgttt gtgtccctgg gtacttgaga ttagggagtg 109680
    gtgatgactc ttaacgagca tgctgccttc aagcatctgt ttaacaaagc acatcttgca 109740
    ccacccttaa tccatttaac cctgagtggt aatagcacat gtttcagaga gcagggggtt 109800
    gggggtaagg ttatagatta acagcatccc aaggcagaag aatttttctt agtacagaac 109860
    aaaatggagt ctcccatgtc tacttctttc tacacagaca cagtaacaat ctgatctctc 109920
    tttcttttcc ccacatttcc cccttttcta ttcgacaaaa ctgccatcgt catcatggcc 109980
    cgttctcaat gagctgttgg gtacacctcc cagacggggt ggcagctggg cagaggggct 110040
    cctcacttcc cagatggggc agccgggcag aggcgccccc cacctcccag acggggcagt 110100
    ggccgggcgg aggcgccccc cacctccctc ccggatgggg cggctggccg ggcgggggct 110160
    gaccccccac ctccctcccg gacggggcgg ctggccgggc gggggctgac cccccacctc 110220
    cctcccagat ggggcggctg gccgggcggg ggctgccccc cacctccctc ccggacgggg 110280
    cggctgccgg gctgaggggc tcctcacttc gcagaccggg cggctgccgg gcggaggggc 110340
    tcctcacttc tcagacgggg cggccgggca gagacgctcc tcacctccca gatggggtgg 110400
    cggtcgggca gagacactcc tcagttccca gacggggtcg cggccgggca gaggcgctcc 110460
    tcccatccca gacggggcgg cggggcagag gtggtcccca catctcagac gatgggctgc 110520
    cgggcagaga cactcctcac ttcctagacg ggatggcagc cgggaagagg tgctcctcac 110580
    ttcccagacg gggcggccgg tcagaggggc tcctcacatc ccagacgatg ggcggctagg 110640
    cagagacgct cctcacttcc cggacggggt ggcggccggg cagaggctgc aatctcggca 110700
    ctttgggagg ccaaggcagg cggctgggaa gtggaggttg tagggagctg agatcacgcc 110760
    actgcactcc agcctgggca acattgagca ttgagtgagc gagactccgt ctgcaatcct 110820
    ggcacctcgg gaggccgagg caggcagatc actcgcggtc aggagctgga gaccagcccg 110880
    gccaacacag cgaaaccccg tctccaccaa aaaatgcaaa aaccagtcag gtgtggcggc 110940
    gtgcgcctgc aatcccaggc actctgcagg ctgaggcagg agaatcaggc agggaggttg 111000
    cagtgagccg agatggcggc agtacagtcc agcctcggct ttcacaactt tggtggcatc 111060
    agagggagac cggggagagg gagagggaga cgagggagag cccctttttt gctttctttt 111120
    ggattatttg aatttttcct taaatttatt tatcttactt atttatttat ttttttgagt 111180
    gattctcctg ccacagctcc caagtagctg ggactgcagg catgtgccac tacacccagc 111240
    taattttttt gtatttttag tagagacagg gtttcaccat attggccagg ctggtcttga 111300
    actcttgacc tcaagtgatc cacctgcctc ggcctcccaa agtgctggga ttacaggcgt 111360
    gagccaccat gccctgcctt tttctagaat ttatatattg agttcttgat tgtatctttt 111420
    tatgtaggct ttttagtggc ttctctagga attacaatat acatactttt cacagtgtac 111480
    tcacatttaa tattttgtaa cttcaagtgg aatgtagaaa acttaaccac cataaaaata 111540
    gaactaggga tgaggttaaa aaagagagag aaaagaaatg taataaagat ttaataacac 111600
    cgtttttttt tttttttctc tttttttttt gagacagagt ctctctttct gttaccaggc 111660
    tggagtgcag tggcgtgatc ttggctcact gcaacctccg cctcctgggt tcaagtgttt 111720
    ctcctgcctc agcctactga gtagctggga ttacaggtgc gcgccaccat gcccagctaa 111780
    tttttgtatt tttagtagag acggtttcac tgtgttggcc aggatggtct cgatttcttg 111840
    accttgtgat tcgctctcct cagcctccca aagtgctggg attacaggcg tgagccaccg 111900
    cgcccggcta agtctttaaa tatttttttg acattgcact ttttctcttt tccttctagg 111960
    attttagtaa cccaaatgtt agttttgtta ttgtttggca ggttcctgag gctttcctta 112020
    cttctttaaa tttttttttc ctgttgttca gcttcgaaaa tttctattca tctgtcttca 112080
    aattcactgg ttctttcccg ttatttccat tctgttattg agtctttgta gtgaatttta 112140
    aattttgttt attatgtttt ttagttctaa aattttcttt ttttgtgtat gtcttatact 112200
    ttgctcctga aactcttatt tgtttcagga gtgatcttat ttcttagagc atggttttag 112260
    tagctactta aaatttgttt tatcatccca gcatatgtgt cctcttgatt gtcttttctc 112320
    ttgtgagata atgggatttt ctggttcttt atatgacaat taattttgga ttgtatcttg 112380
    gacagtttga cttacgttac atgattctga atcttgttta aatcctgtgg aaaatattga 112440
    agtttttgct ttaacaagca gttgacctag ttaggttcag tccacaaatt ctaagcagca 112500
    ttctgtcggc tctggttcca tcatcagttc agttttgtat cttatctgct tatgtgcctt 112560
    tctgtgtcca gtctgggacc tggccaatgg tcaggtccca aagcctttgt acacttttag 112620
    aagcagggcc atgcacaccc agctcacgag tggccccggg agtgcacata caactcgacg 112680
    ttttcatggg ctccttcttt tctgtgatgt ccctgacacg ttctgccttc taagaacctc 112740
    cctttatccc tttcctgttg tctggctaga aagtcagggc tttagattcc ctatacttca 112800
    gcacacttcc tgtagctatg tcaacctctg tggccacgac ttcttcttct tgggactgca 112860
    gtttctcttg tcagaaagta ggattcttgg agctgctgtc attgctgctg tggctgctct 112920
    gatgctgcct gggagtcgaa ggagagaaag gaacaaaaca aaacaaccca ggggatttcc 112980
    tccactctct ttgatccgtg agagccccct ttcctgttcc tcagaccaga aatagagggc 113040
    ctgtcttgga acttcttctt tgtgcatctg gtgtgcagtt tcagcttttg agtccaggcc 113100
    aggaggtgct ggacaaactt gtcaggagta cggaggtact gcaagttctg attacttttc 113160
    tcagtccacc tgcttccaag tccttggatg catttgtcca ttgttttgag ttgcattcca 113220
    tgggagagac agaagagtgt gcttatttca tcttgacata cttattagga tttcatatca 113280
    aatcaacgga tgatattctc tatattaatt tgctgttttc cctttagcaa gcacattagg 113340
    aaaataacac tttaacaccc gcctttggtg gtttctgtca taattattaa tacttgactt 113400
    tttttttttt tttgagacgg agtctcactc tgtcctttga ggcattgtcc ccataaactt 113460
    ttggtaaagc atcaataatt ttatctttca tccacacaag cttcaccata aatttgatgt 113520
    ttattcttcc attttagcag aattcatgtt gctccaatag gggctgtctt caaactgatg 113580
    ttttctcctt cttagtgcct cagagtagat cctgttcaga tacgttataa caggttaata 113640
    tgagtttatt ttggtgtaaa agtactttga aattcatgca tagttttttc atcatatgca 113700
    ttttccatag ctttgaacac ccccatgtaa ctctcctctt ccacaaacca aacaatgaaa 113760
    aagcaccttt gtgatggaag tttattttgc aataggaact cacagtgatc taagccctgc 113820
    tattcatgaa tataattcat tactggagtc caagttgctt tttggttttt gaagttctct 113880
    tcttcccttg caggtataga acaagatgca gtgaatactt ttaccaaata tatatctcca 113940
    gatgctgcta aaccaatacc aattacagaa gcaatgagaa atgacatcat aggtaagcag 114000
    tgcttgaaac tatggcaaaa aaaaaatgac aaaaaatgca cagaactgac aattttcgtt 114060
    attgactaag ataatttttt cttaacatgg aatttagcag ttcccttcct aatttgtttt 114120
    ctgagtattt tttatatcgg attatagctc actttaaaag tttctcggct gcattcggtg 114180
    cgagggtctt tgcctgggcc agatgggctg cagtgtagcg ggtgctcagg cctgcccgct 114240
    gctgagcagc cgggccggcg ggcggctacg ctaaccggca cagaccaccg gatggactgg 114300
    ccggcagccc cgcaccagtg cacgaagtgg gcgggacaga aacttctggg gttggaagtc 114360
    cagtgaggct aaaagccggt accaaagtct ctaggcatca gggctgcagc ccaagagtct 114420
    cacgaccagt gggcaactgg atggccagac aggtgtctca gtggtggcct ctccgtctca 114480
    gggcttcatc ccacttctca gtgggcctga cgtccctggg caccctggat gtctacctgc 114540
    attagccaga gccatcacat ggcctgtgac ttgccttttt ttgccagttg attgtgccac 114600
    acacagtgtc atttctgtgt catttggcac agctggaggt gcaaggagga gggcagcctc 114660
    atgtccagtc ccagtttcac gtaactttat tcttctgaat aaagacaatt tgctaacctt 114720
    aaaaaaaaaa aaaaaaaaaa agtttttctt atatgttgga cccaaattct taggctttaa 114780
    cctgaataac aatgacagca agatcaataa atagtacaca tttattaaac actcactgtg 114840
    tcccagacaa tattccaagc actttttatg gatagactca ttttaacttc taaagaactt 114900
    tgtgggataa atacagttat tttatagatg aagaaactga agcacagaga agttaagtgc 114960
    tttgtccagg gtaacagctc agatatggca gagtcaggat ttgaaactag accctcacat 115020
    accttaactg ctgtgctgtg gcagtgtttt tcatactgta ggttgggacc agccttctct 115080
    tatgccctca ccccctgcca aaaaaaaaaa aaaaaaaaaa aaatatatat atatatatat 115140
    atatatatat atatatatat aatatatata tatataaaat atatatatat ataaaatata 115200
    tgtattagta tatatgcata tatagtatat attatatatt agtatatata ctaatatata 115260
    atatacatat tagtgtgtgt atatatatat atactagaat aaaaaaatca aagtatctca 115320
    gagtagtaag gacaaacatt tcagaaaaat gttttcatta tatatacatg tatgtatgtg 115380
    tatgctgatt caacaaatat atttcttata ggttatagca aaatagtttg aaagctttta 115440
    ctgtgtttta tcaggaagac cttaggtgaa cgtatattca cagataaaag aggttattta 115500
    ttcattcaat aaatattaca ttctcataag tcctaatatt atgtattttt attcttcaaa 115560
    aaagttagta tttgtgattt atgaaataag acatgttctt gcacttttag cagatctgtc 115620
    ccgatgttgg gcttctttaa tccttagtgt gggtgctttg cactcactca ctgctgggga 115680
    cagcaagacc cctgttagtc tcagctgtgt ttcttaaatt ggcccactgt accttccagt 115740
    tagctattct ggggtccatg tcatgttggc tccattttcc ttttctttct cccacacaga 115800
    tacctataac ggctataaca taggcctggt ggctgttggt ggcttatccc tatctgcttg 115860
    tatttaaggg gtactgtttc actgagtttt gctgacagat gttgtcatga gatttgaggt 115920
    tttctgtgtt gttgctctat ttttatgtgg gaatttgcta ctatcatcat ccctagacca 115980
    gcttttccta gtaatacaac agggatgttc tgactgatta gagtttgcct gtttgaagaa 116040
    ttggttggct agtgattttt ttttgagggg agtctgtacc agttaatagc ctgactggcg 116100
    tgtggataaa aaggaagcag tttcaagtca aataaaacac ttaaaatgaa accacactgc 116160
    aactctcttt cttttactta agcttaatca aattaatgat gatgtaatcc catgaaggaa 116220
    aagtcttctg aaggatcaag ttgataacat tttgtgatca aagaatttga gaaaacctct 116280
    atcccagtgt ctatcattat atattttagg atgttaatta cctgtgtggc tttaggcaag 116340
    tcatttttcc tccttgagcc ccattcttaa tcctgtccaa attatttgtc tcctcttgca 116400
    gttggactat tttaatatag ctgtccttca agtgagtttt gttcaaagga gccttcactt 116460
    tagctcttac tgtgtaccca ctttgcatag tcttgtttta aatgtaatcc ttggattttt 116520
    ggtgttgcta actaattact gtttttatgt gaggatttag agtgatccag aatctatact 116580
    tgcactacct ccttcatctt ccacaaatgt ttgaagtggt agaattttta aaaactttga 116640
    aggtacagct gacagaattt gctgatggtt tggaagtgag tggtatgaga gggaaaaaaa 116700
    ggaataaagc atgactgcat tttttgtttg tttgtttgtt tgtttttgag acggagtctc 116760
    actctcgcca ggctggagtg cagtggcgtg atcttggctc acggcaacct ccgcctcctg 116820
    ggttcaagcg attcccctgc ctcagcctcc caagtagctg ggactacagg cgctcgccac 116880
    cacgcctggc taattttttt ttttgtattt tagtagaaac ggggtttcac cgtgttggcc 116940
    aggatggtct ccatctcctg acctcatgat ctactcacct tggcctccca aagtgctgag 117000
    gttacaggca tatatataag catataaagt gtgttatagc atacaaacag gtatatatat 117060
    aaacatgcag tccacacagc tgataggaat gaggcagtag tgaaggagaa gttgatgtag 117120
    gagaggggac agttgttaca ggaaagaagt ctggaggcag aagggatgaa ttccagtgct 117180
    cacatagaag attgcttaga tgggagcaag gacaatttat ctagagtcac aggaaagaat 117240
    gcagtacacg ggtagagatg caggtgagtt gaaagatgtg agagatgatg gaaataattt 117300
    tctgattgct tctatattct caaggaagca ggaagcaaag tcctcagcaa agagaataga 117360
    agaggtgtta aatatttgag aaaggagatg tactgtagaa aaaaaaaaaa ctcagtttct 117420
    ccttctgaac tctcacaaaa cagaaccctt ccatgactct agttgtgtgg ggttttttcc 117480
    ctgtcagcta ccaattctgc agatgattgt tcagtgaaca ccaactgggt gtcctctaag 117540
    tcagttcagt tctcacactg tttacctgga gatagcatca gatcccacag attgaggact 117600
    ctgtcccaca agactgcctc cacttcagat gccagtctca agtacaagtt gtggcctgtg 117660
    cttctgactg accttctata aattggagtt cccacagtcc cctccttggg ttcaataaat 117720
    ttgctagagc agctctcaga actcagggaa atgctttaca tatatttacc catttattat 117780
    aaaggatatt acaaaggata cagattgaac aggcagatgg aagagatgca tgggcaaggt 117840
    atgggagagg ggcacagagc ttccatgcac tctccaggtc atgccaccct ccaagaacct 117900
    ctacagattt agctattcag aagcccccct ccccattctg tccttttggg ttttttgtgg 117960
    agacttcatt atataggcat gattgatcat tggctattgg tgatcagctc aaccttcagc 118020
    cccctcatcc cgggaggttg gtgggtaggg ctgaaagtcc caaacgtgta attctgcctt 118080
    ggtctttctg gtgattagcc ctcatcctaa agctctttag aggccacagc cacaagtcat 118140
    ctcattagcc ttcaaaagaa tccagagatt ccatgaattt taggcgctgt atgctaagaa 118200
    actggctaaa ggccagttgc aatgtctcag gcctgtaatc ccagcacttt gggaggctga 118260
    ggcaggagga tcgtttcagg ccatgagatc aaaaccagcc tggtcaacat agtgagaccc 118320
    ccttacaaaa aatttaaaaa ttggccaggc gtaatagctc ttgtctgtag tctcagctac 118380
    tcagaaggct gaggatcact gagccctgga gttgaaggca gcagtgagcc atgatcgtgc 118440
    cactgactcc ggcttgggtg acaaagtgag accttgtctc agaagaaaaa ggaaaaaaaa 118500
    aaaactgggc aaagactaaa taacatattt cacagtatca cagatttgta ttgtctagga 118560
    aagtgaatgt aaacagacca ggacactagt atgatccctt ggtttcatga aggtcccact 118620
    aaagtcatga acacaaagtg agactaggca tcatgttata tggtttttcc agccatgttt 118680
    aacagctagc taaatagcta attgtttcgc tgcagtttat tttagcagtt ccttatttta 118740
    gcacatttca tgttttaaaa tttctaccaa taacatttta ataaactttt ttacagataa 118800
    cttcacaaat ccataatttt ttaagttaca atcccagaaa tagaattgct cattgaaagg 118860
    gtatgttcat ttttaaagtt atgctagaaa ctgccaaatt gccttcagaa aaaggtgttt 118920
    gtatccccac taacactagt gttagttttc ttgtgccctt gctcaagtat acatattatt 118980
    aaaaacaatg ttgggccagt ttactagata aaaggtgtag tgcctcctta ttctaatcta 119040
    tttgattact agtgagtatg tatgtctttt cacgttggtc attttatgtt tgttcctttg 119100
    tggattgtca tgtcctttgc tcatttttct tttggaacat ttcttagtag tttataagag 119160
    ctcttggtat tttaatgata gtaacctttt aactgtcatg catgctgcaa atcttttttc 119220
    tgtttgtttg cctttgtatt ttgtttttgg agggtttcta tgtataggaa ttaaatttta 119280
    tgttgttaaa tcttttgatt tctgcttttg catatgtact tcaaaagact ttctatttta 119340
    agatcaagtg ttacctgtat tttcttttag ttctatttaa aacctcttaa tttatatgcc 119400
    tgtgctgtta actcccaagt tgattcacaa gtgtgtatac atagtttgaa tttagtggca 119460
    atttaattat ttacaacttc ttttgcagca aggatttgtg gagaagatgg acaggtggat 119520
    cccaactgtt tcgttttggc acagtccata gtctttagtg caatggagca agagtaagtt 119580
    agttcatatt ttcacattgt gcatcctagg gaatttgggt tcattgttag gaatgggctt 119640
    cactcagcta aaaacaaagt atttttgaga atttaaatat tttggatatt tacaagatca 119700
    tataaagcat actctatctt ggttaacagt ttcttttaaa tataaattat gtgaactctt 119760
    aaaattttca ttttcatttt caatgttaat atttcctaag ttaaaataat ttgtttttag 119820
    ttctgaaata atttggggag tgattgagtc tgtagtgatt atgactatta gaattggttt 119880
    atttatttaa ataatgcatg tcttcagatg gctctcctaa tttgttagtt aggctttaag 119940
    ctaaatggat gctatataac taaatccaca tagatttgtt gaaatggctc cagaggtttt 120000
    ttagatttat tactgctatg tgcccttaaa aaaaatctat tcattctttc acttaacatt 120060
    tatcagaaga gtgctctgtg taagacgtgg ttaggcatag tgccagtctt gaaggaagtt 120120
    acagcctaat aaaagacata gggcatgttg tttggttact gtaatatgaa gtggcatgtg 120180
    ttaaatgtca ggggagaact acaaagtcat aaaaaggtgg gagagattac atacaggtaa 120240
    aggaatcagg aatgacacca tggggagtaa ggtagtgttg acctaggcct ttaagataca 120300
    atagggacag tatggaaaga gtatattttt cccacttaaa ctctttcctt ggtcgttccc 120360
    tcaaattttc ccttttgtcc atgtgcaggc actttagtga gtttctgcga agtcaccatt 120420
    tctgtaaata ccagattgaa gtgctgacca gtggaactgt ttacctggct gacattctct 120480
    tctgtgagtc agccctcttt tatttctctg aggtaaagtc tgcatttctt ttcacactct 120540
    attcgagcat tccagcctct aactatcaat gctggggccc tgtctatagg aaataacaca 120600
    gaagagccaa gtcatttcca aaaagatgta tcattgtttc aagttgtttc tgatggcaag 120660
    agtaatttaa taatatatta gagagaacat gaaaattcaa tgtattaaat aactctaatt 120720
    ttgagaaacc taattaaact actgcatgta agagagtgca tgtttttaat tatttggagc 120780
    tattttaaaa ccacagaatt tgaaacttgc ttccagtgca taaattgcag accagacttc 120840
    agaagagaaa aaaagtagta aattttttct tatgctcatc atttttactt tagtcacttg 120900
    ataggattgc ccagtgaaga agcatttgca acagacaatg agtatattaa tctttttgag 120960
    gcatacagtt tagtataatg ctctttgtta ggcttcaaca agtgaaatta ttttgttgga 121020
    aagcaaatga ctattaagta gaaagaggat tcccagtctc acaaagcagt aatttagaca 121080
    ctcgattctg cctctttaca agaatacagg tactcagttg atttgttttc tcactccctt 121140
    tctttgctat aagtttaaat caacaatttg tttaggttaa tatgtcctca tggaatggtg 121200
    gaaatgatca gatataaaat atttggtttg gttagtttac tctttatatg tttgctggca 121260
    aggaaccaca aatccagttt agtataattt ttactctagt tcactaaaag tttgcatcca 121320
    gctgtgtagg tagtgtttgt ttcttgttaa cttttttttc gtctaaaaga atactttaaa 121380
    acttttcaat ctcaaatgac tgtaacttgc tgacaggtgt taacagaaga agtagatctt 121440
    tttgtttttt gcttatgacc tgtattttaa tatttgagct tatagattag agattgtgag 121500
    agaaatctgt ttatagtctt attttccctt gtgtattttt tcttcctagt acatggaaaa 121560
    agaggatgca gtgaatatct tacaattctg gttggcagca gataacttcc agtctcagct 121620
    tgctgccaaa aagggccaat atgatggaca ggaggcacag aatgatgcca tgattttata 121680
    tgacaagtga gttatattga tagatggatt cagcagatac ttattgaaca tttgatatgt 121740
    tttgtggaaa taaagatgaa taaactcagt ctctgttgtc aaggagctca caggaggcag 121800
    cataaaagct gcttttatat ggtgtttgta aagctttggg ggttcttaga acaaaagttt 121860
    ctgctgggaa aggggaggtg tatgtggggt aaacaggatg gcaatggtgg tgttcaagga 121920
    gtgtttccca gaagagagat tttgtttgga tcccaaagaa agaagggaat tttgctaccc 121980
    agagaaggca gaaaacaaca ttctaggcaa aggcattggc ccagaagcca tggaaacgta 122040
    ggggaaagtg gcactttcaa gaaacttgag tttagataat caaaggagtg gggaataaat 122100
    atgaggatgc tggtactaat tggaatagat tgtaagggac cttgaatgcc tatttatggg 122160
    tatattatac tttctgtata aatctgctca ggcacgttgt taattagttt tttattagtt 122220
    ttcactgaaa atgagaggat ggaaacatca tacagtaaac aaaattgaaa atatctggtc 122280
    aggcagatga tgagcttgtg gccagctctg taacgtatgg tattcttttc atttaacttt 122340
    tcttactctg taaaaaaagt aattcgtggt cgggcacggt ggctcactcc tgtaatcaca 122400
    acactttgag aggcagaggc aggtgaatcg cttgagccca ggaatttgag accagcctgg 122460
    gcaacatggc aaaacccgcc tttactaaaa atacaaaaat tagctgagcg tgatggcgtg 122520
    cgcctgttgt cctagctact taggggcctg aggcagaagg atcacctgag ccttgggagg 122580
    tcgaggctgc agtgagctgt gatccactgt actccaccct gggcagggca gtagagtgag 122640
    accctgtctc caaaaaaaaa aaaaacaaca aaggtaattt gttatttgta tccttaagca 122700
    aatgctaaag gggtaacttg gggatagaga aaagtccaca gatgttaggg tttgaagaca 122760
    ctaatagtat ctaggccagt ggttcctgaa cattagtctg tgggctcttg ctgggctgtc 122820
    tgcataggaa tcacctgaga gcttattaaa aataggtttt caggctggtt gcggtggctc 122880
    acgcctataa tcccagcact ttgggaggct gaggcaggcg gattacttga ggtcaggcgt 122940
    tcaagaccag cctggccaac atggtaaaac cccgtctcta ctaaaaatac aagaattagc 123000
    caggcatgat ggcacacacc tgtaatccca gctactcagg aggctgagga aggagaattg 123060
    ctcgagcccg ggaggtggag gttgcagtga gcggagatca tgccactgca ctccaggctg 123120
    gctgacagag ggagactctg tctcagaaaa aaaaaaaaaa ataggttttc agtctgggta 123180
    ccggtggctc acacctgtaa tcccagcact ttgggaggcc aaggcaggca gatcacttga 123240
    ggtcaggagt ttgagaactg cctggccaac atagtgaaac cttgtctcta ctagaaacta 123300
    caaaaaatta actgggcatt ttgacgggtg cctataatcc cagctactag ggaggctgag 123360
    gcaggagaat tgcttgaacc cgggaggcag aggactgcat ctcaaaaaaa aaaaaaaaaa 123420
    aaaggtttcc agtccccctg tctcagaaat tctgattctg caggtttgag gtgtgaccag 123480
    gaatctttat ttttagaaga cataccagat aattctgata aatagccagt ttagggatgt 123540
    agtctaattt tcctattttg caagtaagga aaataaggcc cagagaggta atgattttct 123600
    caaagtcaca gaacaagtta gtggcagaat ttggactgga atgcagttct taatgttctg 123660
    tccagtgttt attctggtac agtatgtttg tagaaggtat tacgtaagaa acattgttat 123720
    atagatgttg agataggaag agtttacatt tagaaatttg gtctaaaatg cctgaacatt 123780
    caagtcgtgg aggagtattg accaacttac tcaatacaac ataggagatt cacattttgt 123840
    tacaaaaatg ctgatttaaa aggagagttt tctttttttt cttctttttt attttttgag 123900
    atggagtctt gctctgtcac ccaggctaga gtgcagtgac acgatctcag ctcactgcaa 123960
    cctccacctc ctgggttcaa gcggttctcc tgcctcagcc tcctgagtag ctgggattac 124020
    aggtgggggc caccacgccc agctaatttt tgtattttta gtagagacag ggtttcacca 124080
    tgttggccag gccggtcttg aactcctgac ctcaagtgat ccacccacca ctgcctccca 124140
    aagtgctggg attataggcg tgagccactg tgcccagcct gcttgttttt gtatcatata 124200
    tatgcatcat cataatcatg cattatcaac ctttgtattt ctgtcaggac atagaaacca 124260
    ttagagtgct tggaagagag cctttttttt tttctcgcat ttaatgcttt ttttggtatt 124320
    catttcataa tcagcttacc aaaacattac ctgcattata ccccatcaag gtagaaatct 124380
    ttgtgttatc aatattggtt actccctttc cacaccgagt catcagtaag tcctgttcta 124440
    tccaaatagg tcatatgcat ctagctcacc cctcagtgct gttttgtttt gaatttgtac 124500
    atgtttactc ctgatgcctt gtagttatga tgatgtgttc ttattttatt ctgtgcatac 124560
    aagttctcag ctcgcttttt agggaaaatg accatgtctt cctttcctat aaattccttt 124620
    ctatctatca agtcctcaac agagaatagg tacccataaa tatgtgattg ttagtttctt 124680
    tgcctcagtt gtagtctgat ccttacagct tttaaacaac agtagagttc accgtcaaga 124740
    actaaggatg gttggcaggc agatagaaag gtagcaagtt gacccaacta tctctgggga 124800
    agtgggaaca aagaaaggtt acatcagcac tgtcatcaca tagctctata gttctaggcc 124860
    tgcaggctca atcaagtagc cttgtataag attctctgga ggaggtgctg aaagttgctt 124920
    atacttgcta tggaatttga ttttacttcg gatatctttt taccataggt acttctccct 124980
    ccaagccaca catcctcttg gatttgatga tgttgtacga ttagaaattg aatccaatat 125040
    ctgcagggaa ggtgggccac tccccaactg tttcacaact ccattacgtc aggcctggac 125100
    aaccatggag aaggtaaccc agaacttcaa acgtatcaaa ctacaagaag ttttattggt 125160
    agaactcata aaatataagg tgggaaaacc aagcagaata gcacagtgga aattgaagca 125220
    gtccagcaaa gtgattaaga gcagaggcct tgagtctggc ctggtatgta cagtcacgtg 125280
    ccacataaca ttttagtcaa cagtggactg cgtgtacgat ggtcctgtac gattataatg 125340
    gatcaaagct ggtagtgcaa taataacaaa agttagaaaa aataaatttt aataagtaaa 125400
    aaagaaaaaa gaaaaactaa aaagataaaa gaataaccaa gaacaaaaca aaaaaaatta 125460
    taatggagct gaaaaatctc tgttgcctca tatttactgt actatacttt taatcattat 125520
    tttagagtgc tccttctact tactaagaaa acagttaact gtaaaacagc ttcagacagg 125580
    tccttcagga ggtttccaga aggaggcatt gttatcaaag gagatgacgg ctccatgcgt 125640
    gttactgccc ctgaagacct tccagtggga caagatgtgg aggtgaaaga aagtgttatt 125700
    gatgatcctg accctgtgta ggcttaggct aatgtgggtg tttgtcttag tttttaacaa 125760
    acaaatttaa aaagaaaaaa aaaattaaaa atagaaaaaa gcttataaaa taaggatata 125820
    atgaaaatat ttttgtacag ctgtatatgt ttgtgtttta agctgttatg acaacagagt 125880
    caaaaagcta aaaaaagtaa aacagttaaa aagttacagt aagctaattt attattaaag 125940
    aaaaaaattt taaataaatt tagtgtagcc taagtgtaca gtgtaagtct acagtagtgt 126000
    acaataatgt gctaggcctt cacattcact taccactcac tcgctgactc acccagagca 126060
    acttccagtc ttgcaagctc cattcatggt aagtgcccta tacagatgta ccatttttta 126120
    tcttttatac tgtattttta ctgtgccttt tctgtatttg tgtttaaata cacaaattct 126180
    taccattgca atagtggcct acgatattca ttatagtaac atgtgataca ggtttgtagc 126240
    ccaaaagcaa taggttgtac catatagcca aggggtgtag taggccatac catctaggtt 126300
    tgtataagta cactctgtga tgttagcaca atggcaagca gcctaacgga aattctgttt 126360
    attgattgat tgattgattg attgattgag acagagtttc actccattgt ccaggctgga 126420
    gtgcagttgc acagtcttgg cacactgcaa cttctgcctc ccaggttcaa ccaattatcc 126480
    tgcctcatcc tcccaagtag ctgggattac aggcaggcac caccatacct ggctaatttt 126540
    tgtattttag tagagacagg gtttcaccat tttggccagg ctgttctcga actcctgacc 126600
    ttaagtgatc tgcctgcttt ggcctccgaa agtgctggga ttacaggcat gagctaccat 126660
    gcctgggcag taactgaaat tctctaatgc cattttcctt atctgtaaag tgacgataat 126720
    atgcacgttt acctcaaagt tactttgatg attaaagtaa ggtaatgtat ataaaataca 126780
    tattaacata gtacctgaca catggtaagc atcaaaaaat gttaactact tttattacta 126840
    ttattattac gtatttttaa ataattagag agcagtatca aaaattagct gggcgtagtg 126900
    gcatgcacct atagttccag ctactcagga ggctgaagct ggaggattgc atgagcctgg 126960
    gaattaaagg ctgcagtgag ccgtgttcat gcccctgcac tccagccttg gtgacagagc 127020
    aagaccctgt cttgaacaat taaagaaggc attatgccgc aacgttagct tagaaatgat 127080
    ccacatatat caccagtaac tgtcaacagg attggaaccc tagttttggg tattatgatc 127140
    acaaggtatt attaatagct tattaataat aaagcgttgg ctaggcacgg cgactcacat 127200
    ctgtaatccc agcactttgg gaggccgagg tgggtggatc acctgaggtc aggagtttga 127260
    gaccagcctg accaacatgg agaaacccca tctctactaa aaatacaaaa ttagccgggc 127320
    gtggtggtgc atgcctgtaa tcccagctac ttaggaggct gaggcaggaa aatctcttga 127380
    acccgggagg cagaggttgc agtgagctga gatcgcacca ttgcactcca gcctgggcaa 127440
    caagagcaaa actccgtctc aaaaatataa ttataataaa taaataaaag taaagtattg 127500
    atgtttgtga atgatttatt cttctaatga actagaggag atttttccag gaatttcaga 127560
    gccagtgagg ttatgttgct tgtatgtgtc atgtgtatcc aggtgaaaaa acttaattaa 127620
    acgctattat ataataccat acataaaaac tgaattttag gaatactgaa gaatgacata 127680
    tagaagtcaa atcattaaat agctagtagt aaacagaata gagtgtcagc tgttacccaa 127740
    tgatgataat attttcacga ttaaaattaa accttttctg attttaaagg aaaagttcag 127800
    atctgtatca tataaagaat gtaaattttc agggtaataa aattaaaatg cagagagaaa 127860
    aatgcaaaaa tagttcttac tagatgtgtg tatgtaagga acttagacta attttaagaa 127920
    cactgtcaag accctggtag ttaggtagga aaaaagacat gaatgattca ttcaacaaaa 127980
    actttgagta tttctgtgct agatggtagt gttacagtgg taaacaaaat aaatgtgttt 128040
    ctgctatcct ggagcttagt ctacaaaaaa ggtacatatt ggccgggcac ggtggctcac 128100
    gcctgtaatc ctagcacttt ggaagatcga ggcgggtgga tcacctgagg tcaggagttc 128160
    aagaccagct tggccaacat ggcgaaaccc cgtctctact aaaaatacaa aaattaactg 128220
    ggtgtggtgg cggacacctg taatcccagc tactcgggag gctgaggcag gagaatcact 128280
    tgaacctggg agacagaggt tccagtgagt cgagatcatg ccactgcatt ccagcccggg 128340
    ggacaaaagc gaaaatacgt ctcaaaaaaa caaaaacaaa caacaaaggc acgtattaaa 128400
    tacgaacata aatatttaca aattatactg aataagttct catgtttatt atttgcttgt 128460
    ccagttacaa acttttcctt cgtagaatta gaaatataaa taataaacat gagaactcat 128520
    tcagtataat taataattat taaatgtaaa taaaaacatc tatgtacaat taggcattta 128580
    tttaagaatt atttgaaaaa aaaacaatgt ggaaacagat attttgatat attgctagtg 128640
    attgaaattg ataatgttct tttgaagagt aaagtgacca tatatattaa agttaaaatt 128700
    taactcagca atcacacgcc tggtgagtta tcttaaggaa atcagtttga aagtaaaatc 128760
    aatatatgca caaagacttt aacatttatc ataaaccaga aaaatcgagt ttcaaattat 128820
    atcctatgga ctattttctg ctaaaaagta ttaatatcaa ctttatgtaa tactttcgtg 128880
    acaaatattt tgggggagaa aacccaacaa aattacatgc attgtaattt tttttttttt 128940
    ttttttttta gacagtcttg ctccagcgtc caggctggag tgcagtggtg caatctcggc 129000
    tcactgcaac ctccatctcc caggttcaag caattctcct gcctcaggcc tcccgagtag 129060
    ctgggattac aggcgctcac caccatgcct agctaatttt tatagttttt agtagagatg 129120
    gggtttcatc atgttggcca ggctggtctt gaactcctgg tctcaagtga tccgtctgcc 129180
    tcggcctcct agagtgctga gattacaggt gtaagccact gcacccagcc ttatgcatta 129240
    taattttaat ttgtaaactg tacaaaggga taatacttgt agtacaacaa gaagtaaaaa 129300
    catttgttat aggtagttaa catttgtaac cagtagaatt ataggtaaaa tttatttatt 129360
    taaaacagtt ttagttggat ttgatttcaa ctttaaaata atgcttttca tctctatcag 129420
    gtctttttgc ctggcttttt gtccagcaat ctttattata aatatttgaa tgatctcatc 129480
    cattcggttc gaggagatga atttctgggc gggaacgtgt cgctgactgc tcctggctct 129540
    gttggccctc ctgatgagtc tcacccaggg agttctgaca gctctgcgtc tcaggtattg 129600
    actgattgcg tctgccatta gggagaaaag catacacatc ctttccttca catcccagta 129660
    acagatccta ttatttgtaa attttaagtt gtggaaaaaa aagataaaag ccaggcacag 129720
    tggcctgtgc ctgtaatccc agcactttgg gaggctgcgg tgggcggatc acacgaggtc 129780
    aggaattcga gaccagcctg gccgacatgg tgaaacccca tctctactaa aaatacaaaa 129840
    attagccggg catggtggca ggcacctgta atcctagcta cttgggaggc tgaggcagga 129900
    gaatcgcttg aacccaggag gcagaggttg caatgaacca aaatcacgcc actgcactcc 129960
    agcctgggtg acaaagtgag actgtgtctc aaaaaaaaaa aaaaaagaga gaaataaaat 130020
    tagcctactt actatcttct aatcaaagca tttgtggtaa cttaaaatat actgtattgt 130080
    aaagtatcat gctgtttcat ttaggccatt attctatttg aatctgtggc tgtttctctt 130140
    aataaatcaa gtaatatgga atatattcat agcctctgaa gagctcttta tgtaagtatt 130200
    tatttaggat actttttgta aaataagtga atgaattctt aggtctcctt tttttttctt 130260
    ttcttgagac agggtctcct cgctgcaacc tggaaattct gggctcaaat aatccaccca 130320
    ccacagcctc ctgaatagct gggactagag gcatgcacca ccacgcctgg ctaatttgaa 130380
    attttttttt ggccaggcat gatggttcac gcctgtaatc ccagcacttt gggagaccga 130440
    ggcaggcaga tcacgaggtc gggagatgga gaccagcctg gccaacgtgg tgaaaccccg 130500
    tctctactaa aaatacaaaa attagctggt tatggtggct catgcctgta atcccagcta 130560
    cttgggaggc tgaggcagga gaatggcttc aaccagggag tcggaggttg cagtgagccg 130620
    agatcacgcc actgcactcc tgcatggtga cagagtgaga ctccatctca aaaaaaattt 130680
    tttttttaaa tgatggagtc ttgctgtgtt gctcaggctg gtcttgaacc cctgacctca 130740
    aatgccgcct gcttcagcct aagtttcttt tttttttgta aagagacagg gtcttgctat 130800
    gttggccagg gtagtctcaa actcctggct tcaagcagtc ctcccacctt ggcctctcaa 130860
    agtgctggga ttacaggcgt gaaccactac ctataatgtt gtgtttcact caaggccttt 130920
    tgatttcgtt ttgcattacc gtgccacatt gtgcatttcc ttgacctttt ttgggttttt 130980
    tggagtgctt tcatatgtta aaccatacct gattctcctc aaaatcacac aaagtagaat 131040
    atcctaagac aagaaatcta aggaggcata aagaagttaa ctggttttat taaactcaca 131100
    cagtaaatga tagagccaga aatattcccc ttctagtgtt cttcaccatc agcttaatgt 131160
    agcataataa ttttctaatt actgttgaca aataaataac cctttgaatt ttcaatactg 131220
    ggccttggat aaattttcct aatttgtaag agagtattat cgtattgcca tttacaaagc 131280
    tctcctgagt atctttttct tctgttaagt ttacctagga gataaactgc tgagtatggt 131340
    tgccattttg gttttttgat ataggttaga atgtcttggt tttttttttt tttttttttg 131400
    gtttttgttg ttgtcattgt ttgagacagc atcttgctct gtcgcccagg ctggagtgca 131460
    atggcacgat cgtggctcac tgcaacctcc acctcccggg ttcaagcaat tctcctgcct 131520
    cagcttcctg agtagctggg attacaggca tgtgcaacca cacctggcta atttttgtgt 131580
    ttttagtaga gaaggggttt caccatgttg gtcaggctgg tattgaactg ctgacctcat 131640
    gatccacctg cctcggcctc ccaaagtgct gggattgcag gcatgagcca ctgcacctgg 131700
    ctgaatgtct tgtttttgat taggcactta agaaaggcct aggtactaac cataaaatat 131760
    atttttatac cttttgttga tactatatat atagaaaact gcacttatca taaccttaga 131820
    caccttgaag aatgttcaca agcagaacta acccatgtga cccagcatcc agatcaaaaa 131880
    cagcattatc agcccctcta gaagccctct tgggcccctt ccattcactg tccttcttgt 131940
    caccagggta gctactatcc tgacttttga tggcatagat tagcattacc tgttcttgtc 132000
    attttataaa taaaaccata ctgtgtattc ttttcttgta cagctttatt gtgctaattc 132060
    acatttacat catacaattc agtggttttt atatggtcac agagttaggt aaccattacc 132120
    acatcgattt tagaacattt ttttcactcc agatagaaac cccctttact taaactccaa 132180
    atcccccact ccaccagccc taggcagcca ctagtctact ttttatctct atagagacaa 132240
    tagatttgct tattctggac atttcataaa catggaaccg tatattatgt ggtcttttgt 132300
    tgccaactgt ctttcactta gcatcatgtg ttcaaaagag catcatgtta tccatgtttg 132360
    gcatgtatca gaattttatt cctcattatg gccaaatatc ccattgcaag gatttatgac 132420
    attttatttg aattgtaccc tcctttctgc catttatcaa taatgctact gtgaccattt 132480
    gtgtacaagt ttttgtgtgg atacaggttt tctttttgtt tttaaatttg aggtggagtc 132540
    ttgctctgtc gcccaggctg gagtgcagtg gcacaatctc ggctcactgc aacctctgtc 132600
    tcctgggttc aagcagttct cctgcctcag cctcccgagt atctgggact ataggcacgc 132660
    accaccacgc ccagctaatt ttttagtaga gatggggttt caccatgttg gccagtctgg 132720
    tctcgaactc ttgacctcaa gtgatccacc catctcggcc tcccaaagtg ctgggattac 132780
    aggggtgagc cactatgccc ggctgtggtt ttcatttctt ttgttgtata tacataggag 132840
    tagaattgct gagtcaagag gtaactctta aacttattga aaaactgcca gattgttttc 132900
    cgaaaaggct gcaccatttt gcaatcccac cagcagtgta tgagttttac agcttctcca 132960
    catttcattg gaacttatta tctgtttggc tgtttttaaa aatgatagtc attccaataa 133020
    gttctacttc agtgtggttt ttgcacttct ctgatgagta atgatgttga gcatcttttc 133080
    atttgcttat tggcctttgt tctagctttg gaaaaatgtt tattcaaatc ctttggccat 133140
    ttttattttt atttttattt atttattttt ttttgagacc aagtctcact ctgtcagcca 133200
    ggctggagta caatggtgtg gtctcagctc actgcaacct ccgcctcctg tgttcaagtg 133260
    attctcctgc ctcagcctcc cgagtagctg ggattacatt tcaggcacct gccagcatgc 133320
    cgggctgatt tttgtatttt tactagtgac agggtttcac catgttagcc aggctggtca 133380
    caaactcctg acctcaggtg atctgcctgc ctaggcttcc caaagtgctg ggattacagg 133440
    cgtgagccat tgggcccagc ctagattttc ttttttcttt ttttttttga gaaggagtct 133500
    tgctcttgtt gcccaggctg gagtgcaatg gcacaatctt ggctcactgc aacctctgcc 133560
    tcctgggttc aagcgatttt cctgcctcag cctccccagt agctgggatt acaggtgcct 133620
    accaccacac ccagctaact tttgtatttt ttttagagac agggtttcac catgttggcc 133680
    aggctggtct caactcctga cctcaggtga tccacctgcc ttggcctccc gaagtgctgg 133740
    gattaccggc atgagctacc aggcccagcc aattttctca ttatattgcc caggctggtc 133800
    tcaaactcct gggttcaagt gatcctcctg ccttggcctc ccaaagtgtg gggagtacag 133860
    gcgtgagcca ccttgctcag cccctttgcc catttttaaa ttagattgcc tttttatatt 133920
    gagtttcagg agtcctttat atattctaga taaatgtccc ttatcaaatt atattatttc 133980
    caggtatttt cttcattctg tgagttgtct ttcctctacc ttttaaaaaa ggtgggtttt 134040
    tgtttgtttg tttgtttgtt tttttaagat aaggtctcat tctgctgccc aggctggagt 134100
    gcagtggcac aatcacagct cactgccacc tcaacttcct gggccgaagt gatcctctta 134160
    cttcagcctc ctgaatagct agggccatag atacacacta tcacacccag cttttttttt 134220
    ctgtttgtag agacagatct tactgtgttg cccaagttgg tctcaaactc taggctcaaa 134280
    gtgattctcc cacctctgcc tcccagagtg ctgggattac aggtgtgagc cacacgcaac 134340
    ctgtcttttc actattaata gtgtcttcct gcttcagcct cccgagtagc tgggattaca 134400
    ggcacccacc accatgcctg gctaattttt ttgcattttt agtagagaca gtgtttcacc 134460
    atgttcaccc ggctggtctt gaactcctga cctcaggtga ttcacctgcc atggcctccc 134520
    aaagtgctgg gattacaggc gtgagccact gcacccggcc aaaatattgc cttcttaaca 134580
    gtattgtctt ctaatttgtg aacatggatg tatcttcatg tatttatgtg ttctttcatt 134640
    tcagcagaat tttgtagttt tcagagtaga agcctttcac ctccttgggt catttattcc 134700
    tatgttttaa gttcttttcg attccattat aaatagaatt gttttcttaa tttcattttc 134760
    agattgtttg atgagagagc atagaaatac aagtgatttt tacatgttga tcttgcaact 134820
    tcaactttga taaatctgat tgttagctct aatagttttc ttgtggattc tttaggattt 134880
    tcaatatata agatcatgtc atttatggat agagatagtt ttttttctgg ctagaactta 134940
    cagagcaatg atgagtagaa gtggcagaag caaaaatctt tgtcttgttt cctatctgac 135000
    agggaaagct ttcagtttca tcatttaata tgatgttagg tgtgggtttt caataaatgc 135060
    cttttttcag attcaggaat ttccctatca ttcctgattt tttaaggctt tttttttttt 135120
    ttaaatcatg aaagggtgtt gaatattgtc atgttctttc tgtatcagta taaatgatcc 135180
    tatggatttt gggttttatt ctgttgatgt gaaatattaa ttgattttca gatgttaaac 135240
    caaccttgca tacctgagat gaatctcact tggtcatggt gtataatctt ttcaatatgc 135300
    tgctggattc catttactgg tattttgttg aagattttgt atctgaacgc ttaagataac 135360
    atttacactc tatcagaaat gaattgacca taaatgtgag agtgtatttg tgggttcttg 135420
    attctcttcc attccaaaga tagacataca tccgtctgta tgtctgtctt tatgccagta 135480
    ccatactctc ttgattacta ttgctttgta ataagttttg aaatcagaaa gtataaatga 135540
    gattttggta tctgagtaac agtcctcata gaattagttg ggaaatattc cctctttatt 135600
    ctggtccctc tttctttttt gtttaactgt gtatcttgga gattgttcct tctcaacaca 135660
    tgagagccgc tttccctacc ctcccacccc tgctatagag aggtctataa gtgtctgttc 135720
    aattatttta tttacttaac ctattactta gtcggggaca ttaagcttgt ttatgtcttt 135780
    tattttaaac aatgctgcag tgaataatct tgtatataag tcattttcca tcaatataag 135840
    tctctctgta actgaatttt tagaagtgga atttctaggt caacctatgg ctctgtattt 135900
    cacaaaaata ccaattctgg tttttcttgt ggaggtgggg agtaggaggt agaatgctgg 135960
    aggagaactt gctgtactca gctggctagt cattttagaa aggtttcctt agcttctttt 136020
    tgtcatatgg cctcaccaag aatcaaaaac attcctattt accctgtaaa catggggctt 136080
    tactacccaa gatacatatt tctggatgta tgacagcttt tcatattgaa gaaataatgc 136140
    tgtgagtaca gcacatttgt tggaacttag gtcgttaaga atgtcttata aattcataca 136200
    ttatacattt tattttattt tattttttag tttttgatac agagtcttcc tctgtcgccc 136260
    aggccagcgt gcagtggtac aatcttggct cactgcgacc tccatctcct gggctcaagt 136320
    gattctcatg tctcagcctc cagagtagct atggttacag gcatgcacca ccatgcccgg 136380
    ctaatttttt tatttttagt agaaactggg tttcaccata ttgaccatgc tggcctcgaa 136440
    ctcttggcct caagtgatcg gcctgcctca gcctcccaaa gtgctgggat ccttgtattg 136500
    ggtaaaagat gaatattgag ggctgcatgg tggctcatac ctgtaatccc agcactttct 136560
    gagactgagg tgggaggagt cctggagccc aggagggtga ggctgcagtg agttgtgatc 136620
    gcgccattgc acttcaacct aggaattata ggcttcagtc actgtgcccg gcatgtacat 136680
    tttaatattg tgctttcctc ttttagctat agtatgaggt tacatttcag agtcattgtt 136740
    gttaagcatc ttaatagtga tgaggttgag tgaaagttac ttctatttca aacactgaag 136800
    aaaattttgt acaaatctgt cacattccaa gcccaggact gattgtttca tatacttcta 136860
    attttacaat ttctattgta gtccagtgtg aaaaaagcca gtattaaaat actgaaaaat 136920
    tttgatgaag cgataattgt ggatgcggca agtctggatc cagaatcttt atatcaacgg 136980
    acatatgccg ggtaagctta gctcatgcct agaattttta caagtgtaaa taactttgca 137040
    tcttttaaat tttttaatta aattttacat ttttttctaa tctattatta tatgcccaga 137100
    actttcactt agagtgtgca gtataatgtg gtggttaagt ataaaggctc tggagtgact 137160
    tcctgggttt taatcttggc tctgccattt attggcagcc gctaacctct tggtatctca 137220
    gtttcttcat ctgtaaaatg agaataataa agtgaaaaga tgccaacatc atttactctg 137280
    ggctgcataa ctgatacttg gaaaaagtat tcctttgagt ttaagaatta agttggttat 137340
    tcattttagc ttgtaataaa aagatagtga ttcataggat atgccactta ctgaaattta 137400
    ccacagatcc aatcataaaa tcactttctc ttccctaaag atagcttgat taacatgtaa 137460
    aggtgtgtaa aggcttgatt acactaccct gatccgtacc ccagttccca gcagcaccat 137520
    gaaaaaggga tttcaacata tttaattact ttcagtagaa agtaacagtg gtaggccagg 137580
    cgcagtggct cacacctgta atcccagcac tttgggaggc cgaggtgggc ggatcacgag 137640
    gtcaggagat tgagaccatc ctggctaaca cgatgaaacc ccgtctctac taaaaataca 137700
    aaaaattagc cgggcatggt ggcaggcacc tgtagtccca gctacttggg aggctgagac 137760
    aggagaatgg cgtgagcccg ggaggcggag cttgcagtga gcttagattg tgccactgca 137820
    ctccagcctg cgcagtggag cgagactctt gtctcaaaaa aaaagaaagt aacagtggta 137880
    ttgggagact gaggagccta gaaagtactt gaaggaagta aaaggtttgt ttgaccacat 137940
    tgtatttgga aagccagctt tttcagctgt gtcagctttg tgtagtgatt tttagttctt 138000
    cttttagaaa ataacggaca aggccgggca cggtggctca cgcctgtaat cccaccactt 138060
    tgggaggccg agacgggcgg attacctgat ctcaggagtt cgagaccagc ctgggcaaca 138120
    tggtgaaacc ccgtctctac taaaatacaa aaagttagcc gggcgtggtg gcgtgtgcct 138180
    gtagtcccag ctactccgga ggctgaggca ggagaattgc ttgaacccgg gaggcggagg 138240
    ttgcagtgag ccaagatcac accattgcac tgcagcctgc gcgacagagt aagactctgt 138300
    ctcaaaaaat aataataaaa taaaaaagaa tggacagtaa acctaaatga gttcattccc 138360
    aaagatgatg ttattcttaa gggatggttc atttatttaa gaccttacat aaagtctatc 138420
    aattgcgtga tttttcactt ctgtaattgt gtgtatgtat aatgtaaata tatatgtttt 138480
    tgttttgttt tggttttttg agacggagtc tcgctctgtt gctcaggctg gaatgcagtg 138540
    gtgcaatctc agctctctgc aacctctgtc tcccaggttc aagcgtttct tctgcctcat 138600
    cctcccaagt agctgggact acaggcacgt gccaccacgc ccggctaatt ttttgtattt 138660
    ttagtagaga tggggtttca ccgtgttagc caggatggtc tcaatctcct gacctcgtga 138720
    tccacccgcc ttggcttccc aaagtgttgc tattacaggc atgagccacc acacccagca 138780
    tgtatttttt aaatgtataa aatgaagcag aaaagagaaa tgataatttt tcttcatctt 138840
    gaaagattat cttcaccagg cgcagtggct cacacttgta atcccagcac tttgggaggc 138900
    ctcggcaggc ggctcacttg agttcgaaac cagcctggcc gacatggtga aactccgtct 138960
    ctactaaaaa taaataaata aagatggttt taatatatgt tttagtttta tgattttagc 139020
    atctttctga aatttttctc aaggcaagta aatttgtatc agttggtata ttggtaccca 139080
    tctatgaaat aacttattag gaagatatct ctaaaataag atcactttgc ctaaaataaa 139140
    ctgatatatt gatgttcaca gaatttttct tttaaccgac ttgataaatg cattattctt 139200
    gacgtcaagt gatccacctt cctcagcctc ccaaagtgct gggattacac acatgagcca 139260
    ccgcacctgg cattattctt ataaaaggtt aaatttctag ttaagtttaa tgtcctcttt 139320
    gttcatgtac cattgcttat tttcttccct tcctactcac agtaatcatt cttatggtat 139380
    gcacttttgt ttgcttattt ttatgtaatt gatattacgc tccattctgt acgttgtact 139440
    ttcattcaca gtgagttttg gacattccta tgttcatcta tacagactta cttcatttta 139500
    actacactgt agtattccgt atgtaatatt tactataact catcactgta gcagagcatc 139560
    tcatagtgta tgtattactg ttttgccatt ttggtatcaa tgagtattta agtcatttgc 139620
    agtttttccc tcttataccc agtattacag aggatctctt tttatatgct tctttgtacc 139680
    aagaggcaga ttaaaaaatt tttttttgaa aaaatttttg aaaaaaaatg aaatgaagtc 139740
    tcactatgtt gcccaggctg gtctcaaact cctaggctca agcaatcctt ccatcttggc 139800
    ctcccaaagt gctggggtta caggcatgag ccaccatgcc tggcctacat tttaaatttt 139860
    gatagctctt acaatttact ttgtaaagta tctgcatcat tttatgttct caccagtctt 139920
    taataagaat acttcatact tttggctgga cacagtggct cacgcctgta atcccagcac 139980
    tttgggaggc cgaggcgggc agatcaagag atcgagacca ccctggccaa tatggtgaaa 140040
    ccctgtctct actaaaaata caaaaattag ctgggcgtgg tggcgcaccc gtagtcccag 140100
    ctactcgaga ggctgagaca ggagaatcac ttgaacccgg gaggtggagg ttgcagtgaa 140160
    cttagatcac accactgcac tccagcctag caacagagtg agactctgtc tcaaaaaaaa 140220
    aaaagaatac ttcagactta attttttttc cagtcttaag tgtttgctaa tgagattgag 140280
    tttcttttgg tatgtctctt gattgttcag gttttttctt ttatgaattg actgttcatc 140340
    tctttttcac attatttctg ttgggtgatt ttattagtga cttgttaaaa ttctgtatat 140400
    tttttcagca tgacacttca ttattcaaaa aaaaaaaaag attctctatg tttctcgata 140460
    ctaatcattg gttggtaata ccttaaaaat aagaccctta ctgtattttt tgcttttttt 140520
    tttttttttt tttttttttt tttgagatag agtcttgctc tgttgcccag gctggagtgc 140580
    aatggtatga tctcggctct cagctcactg caactgcaac ctctacctcc ctgtttcaag 140640
    caattctcct gccttagcct cccaagtagc tgggattaca ggcatccacc accacaccca 140700
    gctaattttt gtatttttag tagagacagg gtttcaccat gttggccagg ctggtctcaa 140760
    actactggcc tcaagtgatc cgcctgcctc ggcatcccaa agtactggga ttacaggcat 140820
    gagccacagt gcctagccac tttttgcttt ttaactttgt tttatagtac tatagtttta 140880
    gtataaacag atgtatgtat acacacaact atggctttat aatatgtttc agtcattgtt 140940
    agagcaaggc ctaccttttg ggtgcttctt ttacaaaatt gtcttggcta ttcttgtgcc 141000
    ttttttctta tttgtgaatt ttagaattgt gaattacctg ttgactcacc atgttttgta 141060
    aactgaggat tttgaatgga attgcactca attaaagatt atcttgcttt ctgtgcagca 141120
    atgttttatt tcaaataatc cctactttaa attacttagg atagctataa attgtgtttc 141180
    tggctttcta gatttagatg aaacgcttta aattgattgt tttctcctaa atttaaaact 141240
    gattgttaga agttaaagtc ttctgttcat tcttatttag gaagatgaca tttggaagag 141300
    tcagtgactt ggggcaattc atccgagaat ctgagcctga acctgatgta aggaaatcaa 141360
    aaggtttgtg gtgtttttat acttcatatt aagcctttac tcacattagt gattgactgt 141420
    aagtcaaaga ccacttaagg tttaaactgt ttattttgta aagtaaccac tgtatctttc 141480
    accttgtgtt tatagtcaga agtaagtaca agggcttcct gtagtcacat ctttatgcaa 141540
    tctcctctga atcaaaagtt agtgaacttg ctttgccact ccagaaggca catgaatatg 141600
    aaaaagcatt gtctattttc ttatttaatg gcaaaatacc cgacctaagt tggacttaat 141660
    gtttgagacc gtttatttta ttaaattata ttttttctct tttctttttt ttttttgaga 141720
    cagttcttgc tctgtcaccc agaccggagt gcagtggtct gaccgcacct cactgcaacc 141780
    tctgcttcct aggttcaagc gattttcctg cctcatcctc ctgagtagct gggactacaa 141840
    gtgcgcacca ccacacctgg ctaatttttg tatttttagc agagatgagg tttcaccacg 141900
    ttggctaggc tggtctcata ctcctgacct caagcaatcc atccgccttg gcttcccaaa 141960
    gtgctgggat tacaagtgtg agccaccatg cctggcctta ttaaattatt tttattaaat 142020
    ttcctcaaga ttgatgaaag taatgaaata taaaagtaat gaaatatatg tggaaaatag 142080
    actggattaa gaaaatgtgg cacatataca ccatggatac tatgcagcca taaaaaagga 142140
    tgagttcatg tcctttgtag ggacatggat gaagctggaa accatcattc tgagcaaact 142200
    gtctcaagga tagaaaacca aacaccgcat gctctcactc ataggtggga attgaacaat 142260
    gagaacactt ggacacaggg tggggaacat cacacgctgg ggcctgtcgt ggggtggggg 142320
    gctgggggag gaatagcatt aggagatata cctaatataa atgacgagtt aatgggtgca 142380
    gcacaccaac atggtacatg tatacatatg taacaaagct gcacgttgtg cacatgtacc 142440
    ctagaactta aagtataata aatttaaaaa aaataaatat atgtggaaaa tattaatagg 142500
    tcaaaattca aattgttcat ttaatcagaa gagtagttta gtcaaatcca agggttagac 142560
    aacagaaatc ttttttgtca agtgcattct ttgtgactga tttcattttc ttcctggttt 142620
    acacaggaag atttcagaaa caaatgtgga tccgtgacag atggtatcta gaagttttta 142680
    gtttggttga attgacagta ttttattgag taaaagatac taatttttgt aagaagaaaa 142740
    attcaatttt gataagtatg tttaagatta agagctattg gccaggcgct gtggctcatg 142800
    cctgtaatcc tagcactttg ggaagctgga gcaggtgggt cacgaggtca agagattgag 142860
    accatcctgg ccaacatggt gaaaccctgt ctctactaaa ttagccaggc gtggtggcac 142920
    atgcctgtgc acccgcctcc gggtttaagc gatcctactg cctcaggctc ctgagtagct 142980
    gggattacag gcgccatggc taatttttgc atttttagta gagacagggt ttcactacat 143040
    tggccaggct ggtctggtct caaactcctg acctcaggtg atctgcccgc cttagcctcc 143100
    caaagtgctg ggattacagg catgattcac catgtctggc catttatctt attttctttt 143160
    tttttttttt ttttgtttga gacggagtct tgctgtgtcg cccagagctg gagtgcaatg 143220
    gtgcgatctc agctcactgc aacctctgcc tcctgggttc aagcaattct cctgcctcag 143280
    tcttccaagt agctgggatt acaggcgcgt gccaccacat ctagctaatt tttgtatttt 143340
    tagtagagac agggtttcac catgttggcc aggctggtct cggaactcct gacctcgtaa 143400
    tctgcccacc tcggcctccc aaagtgctga gattacaagt gtgagccact gtgcccagcc 143460
    atcttatttt ctttcttttt ttttgtcggg tgggaggggg acagagtcta gctctgtcgc 143520
    caggcttggc tcactgcaac ctctgccccc caggttctag caattattct gcctcagcct 143580
    cccaagtagc tgggattata ggcacctgcc accacgcctg gctaattttt tgttattttt 143640
    agtagagatg gggttttgct atgttgacca tgctggcctc aagtgatccg cccaccttgg 143700
    cctcccaaag tactgggctt acaggcgtga gcttgtattg ggtaaaagaa caatattggg 143760
    ggctgcatgg tggttcatac ctgtaatctg agcactttgt gagactgaga tggaaggagt 143820
    gttggagccc aggagggtga ggctgcggct gcagtgaatt gtgatcacgc cattgcactt 143880
    ccacctaggt aatggagcaa gaccatgtct ctaaaaaaca aaacacaatt tttttaagga 143940
    atactgggaa gaggtcagtg gtggttttag aacagaggaa gtgccagatg acctttgtga 144000
    ggcattggcc aggaagaact ctacagtgtc tttaggtagc ttctgtccat aaggataatg 144060
    gggtctcctc cccagtatta atagaaaatc tctgagctgt ttttttttgt ttgtttgttt 144120
    tgtttttttt tcctgagatg gagtctctct ctgtcggcca ggctggagtg ctgtggcgcg 144180
    atcttggctc actgcaagct ctgcctccca ggttcacacc attctcctgc ctcagcctcc 144240
    caagtagctg ggactacagg tgtccaccac cacgcccagc taattttttg ttatttttag 144300
    tagagatggg gtttcaccat gtcagccagg atggtctcga tctcctgacc tcgtgatccg 144360
    ctcgcctctg ccttgcaaag tgctggagtt acaggcgtga gccaccgtgc ctggcctggt 144420
    ttttttgttg ttgttattta tttatttatt tatttatttt ttgagacaga ctctcgctct 144480
    gtcgcccggg ctggagtgta gtggcacgat gtcggctcac tgcaagctct gcctgccagg 144540
    ttcaagccat tctcctgcct cagcctcctg agtagcaggg accacaggcg ctcgccacca 144600
    cgcccggcta attttttgta tttttagaag agacggggtt tcaccgcatt agccaggatg 144660
    gtctcgatct cctgatgtcg tgatccgccc acctcggcct cccaaagtgc tgggattaca 144720
    ggtgtgagcc accgtgcctg gcctgatttt tttttttttt taatctggtc tcatacctct 144780
    gacagctcat gaagaagtgc tcctgcttca tatgtatatg tgttagcata gtgttaacat 144840
    agcataggtg ttcggtgttt gcagtttctg tttgttttat atgaattaag gtgtattatg 144900
    agcagttgaa gatatatagg aaattttttc ccaaaccact atctctgctc gttctattca 144960
    ttcagtctgt ttatgttatt ccttcattca ttcattttat agaacagtgg agtgcctact 145020
    gtatgcatct attgttctgg gtcctgggga agaaaacaaa gttcctgctt tcatggaact 145080
    tacattatat tggcggagac agtaacagac aaacaaatgt agcctgtgta catgtgttac 145140
    atgaaaagca gggtaggggg ctgggagaga gtagtaggga gtgctatttt cgaggtggtt 145200
    gtcaggaaag gcctcactga ggaggtggca ttttgagtag acctgagcgc agcgggggcg 145260
    taagcccagg cagcatgtgg aggaagagtg ttcttggtga aaggaacaag gatagaggcc 145320
    cgaagctaga gagctcagca tgatcaagga acagcaagcc ccgtgtggct ggaatggagt 145380
    gagcaaagga atgagcagta gaaggtgagt gagttgggag gtcaccagag accatggcaa 145440
    ggacttgaaa gtgtcaggga cacattggaa gttggagcag ggaaatgatg ggatttatgt 145500
    tttgtttttg ttttatgttt agtgttttta agggattgct ctatcagcta tttggaaaat 145560
    ttagtgtagg gcttcaagaa gagaagcaga gaaacaacat tcttgccata gtcatagtct 145620
    aagtaaggga tgatggtggt gtggattagg ctggtagtgg aagaccagtc cagttcgggt 145680
    tgtatttgaa ggtagaggca aaaagattat atttctacca gcaagcccat ctatgaagtt 145740
    acttgtatta ttaatttaat tgagacatgc ccacataaac taataaatag gaatttctgc 145800
    agtttggtta aacacccctg tatatcctgg ttcttctttt agttgtccag atgtctcttt 145860
    aagtcaagta ttttttggtg gtgtaggagc ctagagattg aatttattca cccaaaaggc 145920
    atttgagtga ttactatgtg ccaggcacta tgctgaatgc caaggatgta aataagaggg 145980
    cgtagtctca gtctgtttta ctccagcttg gttccttttt aatgaccctg acttgttaag 146040
    catatcagtt atcctacaga atgtttaatc ttctgtactt tcctggttgt gttatttagc 146100
    ttatttctct ttccttgaca tttcttgtaa actggaagtt acacctatag tcttgatgat 146160
    tcgtgttaca cattttagat tagaacacat catgtgttgt atatggtgtt tttgaaagcc 146220
    tctctgtata ttggtctgta cattaaaatg ttgcctgaat ggatacacat aaaatttaac 146280
    agtgattaca ttagagatga gaagaaagag gtgcctttta cttttcaata taccttttcc 146340
    tctgcttttt gaactttctt gccctatgca tacgttattg cttaatcatc cacctcatct 146400
    cttcccctgt ggctttctgt tgcatttgga atgaaatcta gcctctttgc tgttacctgt 146460
    ggatgtccct tgctggcctc tatcacctta ctttgaacca ctcctttcat ggactgagct 146520
    ctcattggac tatcttttat tcttttgctg aagtttcttc actttgagtg cctctgcagt 146580
    tgctatttca tggctgtggc aagccctgcc atggctttca tgcaaggatg gttcctcctt 146640
    ctcatctcaa tattatctct tcagagaggg accttcccaa ctccgatgat ctaaaatcct 146700
    ttgtatatac cactcactac cacttctttc ttttcttttc cttttatctt tttttttttt 146760
    tttttttttt gagatagggt cttgctctgt tgcccaggct ggaatcacga ctcactgcag 146820
    cctcatcttc ttgggctcaa atgatcctct cacctcagcc tctcgagtag ctggaactgc 146880
    aggcacacac caccatactt ggcttattat tttacttttt gtagagacag ggtttcacca 146940
    aggctggtct caagctcctg ccgcaagcaa tccacatctc tcagcctccc aaagtattgg 147000
    gattatagga gtgagccact actcctggcc tattttctta ttcactgtct aaaattatct 147060
    tgttcattta tttacatact tgtttatagc ttatttctca gctggacatg gtgcctcaca 147120
    cctgtaatct caatactttg ggaggctggg ttggagaatt ggttgagccc aggacttcaa 147180
    gaccagcctg ggcaacaaag tgagaccctg tctataaaaa attgtttaaa aattagctgg 147240
    gcatggtggc acatgcctgt ggtcccagct acttgggagg cagaggtggg agaatcgctt 147300
    gggcccagga ggttgaggcg acggtgagcc atgattgtgc cactgcactc tagcctagtg 147360
    acagagtgag accatgtgtc taaaaagtaa ataaaaatag tttctctttc atgactagaa 147420
    tattacctct atgtgggcag ggagtttgtc tatactattt ggcactatat ttcctgattc 147480
    tgaaattatg cctagcacat ggtaagtact ccttaaatat ttattgactg aattatttaa 147540
    tacttaagaa tttcatttgg gattatctga gtggtaagat tacggattat atttatgtaa 147600
    gaaaaaatca ttttttaaac ttggttgccc tttgccacac tgacatagac actaagtttt 147660
    cttagccaga ttacttccga ggatactcac agaggccatt ctcttctcaa tccccaaata 147720
    attgatattt cttagcactt tcaagctaat gcaattctta gatgatgtat ctgtgtatat 147780
    catatcctca ttctacaaat gtagaaattg aagtctgggc acagtggctc tcacctgtaa 147840
    tctcagcagt ttgggaggcc aaggcgagcg gatcactgag gacaagagtt aagaccagcc 147900
    tggccaacat ggtaaagcct tgcctctatt aaaaatacaa caattagggc cgggcgtggt 147960
    ggctcacgcc tataatccca gcacgttggg aggccaaggc aggcagatca cgaggtcagg 148020
    agttcgagac catcctggct aacacagtga aaccccatct ctactaaaaa tacaaaaaat 148080
    tagccaggca tggtggcacg cgcttgtagt cccagctatc gggaggctga ggcaggtgaa 148140
    tcccttgaac ccgggaggcg gaggttgcaa tgagctgaga ttgcaccgct gaactccagc 148200
    ctggtcaaca gagggagact ctgtctcaaa aaaaaaaaaa aaaaacaatt agccaggcgt 148260
    ggtggcgggt acgagtacct gtaatcccag ctactaggga ggctgaggga ggagaatcac 148320
    ttaaacccag gaggtggagt ttgcagcggg ctgataatgc accactacat tccagcctgg 148380
    gcaacagagt gagactctgt cttaaaaaaa aaaaaaagaa agaaagaaat tgaggaatgt 148440
    ggagattgtg gtctgtgatt tgttaggaat cacacagcag gttagtagca actacagggc 148500
    tttggttcag aataccacct tgacaatggt ttgtttacag ttcggctccc cttcctctgc 148560
    ctttctctcc ttccttattg agggcagctg gaaagaattt tcatcattta ctagcctata 148620
    gctttaattt gagttttgaa accttgataa tagagcacag aggaaaagac tgagttttct 148680
    ttttttgaga cagtcttgct ctatggccca ggctggagtg cagtgacacc atctcagctg 148740
    gttgcaacct ctgcctccca ggttcaagca attctgcctc agcctctcga gtagctgaga 148800
    ttacaggcac gtgtcaccac gcccagctaa ttttctgttt ttgtttcgtt ttgttttttt 148860
    ctgagatgga gtcttgctct gtcacccagg ctggagtgca gtggtgcgat gttggctcac 148920
    tcaaacctct gtctcctggg ttcaagcaat tcttctgcct cagcctcccc agtagctggg 148980
    actacaggta cgtgccacca tccctagttc atttttgtat gtttagtaga gatggggttt 149040
    cactatgttg accaggctgg tctcgaactc ctgatctcag gtgatctact cgtctcagtt 149100
    tcccaaagtg ctgggattat tggcacacgc ctatttttgt atttttagta gagacggggt 149160
    ttcaccatgt tggttagact ggtctcaaac ttctgacctc aagtgatttg cccgccccag 149220
    cctcccaaag tgctgggatt acaggcgtga gccaccgtgc ccagccaaga ttgagttttg 149280
    aaaagagcct tctgagatta tgagaagggc aagcaagata acttaagaag ttacattaaa 149340
    atcatctaag agacagtgta acaagaagga attgtaaaat gatgttatga gcacgtgccc 149400
    aatgtagtgg caatcccttg tgcttcgata cattggtggg agacaaaact gtacttaaat 149460
    tgataaatcc cttacatgtc attttaagga gcttagactg actcccatca tgtagacatc 149520
    agagatttct tttttttttt tttttttttt tttttttttt tttgtgacag agttttgctc 149580
    ttgttgccga ggctggagtg caatggcgtg atctcggctc accacaacct ccacctccca 149640
    ggttcaagca attctcctgc ctcagcctcc cgagtagctg ggattacagc catgcaccac 149700
    cacgcctggc taattttgta tttttagtag agacggggtt tctccatgtt gtggctggtc 149760
    tcgaactcct gacctcaggt gatcctcccg cctcagccac ccaaagttct gaaattacag 149820
    gcgtgagcca ccgcgcccag cccagagatt tctaaacaga gttctaacca gatgcttttc 149880
    cctgtcagta gaatgagaat gaattggagg tgggagagac tggcatgagg gacaccagtc 149940
    agccagtgga attagctggt aatgttgata ggagaagaaa aagattcaaa gttaggtagt 150000
    ggtagcaaga attagaggga aggtcggatt tatgatatgt ccaaggttga attctaaggt 150060
    gaaatttggt ggcagatttc atgtgtaaat tgggaaggta gattgagttt ttttaacatg 150120
    ggttttctaa catgtcaata gagtgactct gcaggggggc ctgacgagag aacagtgcat 150180
    ggggtgattc aacagccagt tgagccttca tgcagagcat ttaacactgt gactctgtag 150240
    actctggttg gcagtaaaat ttcattaaac caatatttaa acccttaggt aataataaaa 150300
    attgagggaa aaggatccag gttttgtatt ttttatgaat tcagttattg aattaaacag 150360
    gaccttgcct caagaaataa tctaccaaca attaacttgt tttaaagcaa agttaggaag 150420
    tgagcatgtt caaattatta aataaaaaag taagctgtgt atttcattca tagaaataga 150480
    ggctggccta cttcggatga ttctcagcat gtgattacag atgtgggctt atacatccta 150540
    gggagttaag gcgtactctg gcttggatag agtagagctc tttgaaactc ttctctcacc 150600
    cagctagttt atatagacta gagaactaga atgtagcagc atactctgtc ttagaagccc 150660
    ttttatatag gagctggtct ggaaggtttg aaaacataac aaatgtgttg gtgtctccca 150720
    atgtattgct agattcttac ccaagagcat tatcctggtt agggtttggt ttggttttgt 150780
    tttgtttttt aatgtttgcc acaaactaac actagatgtt agttctttca tcaagtgagg 150840
    agagtagaag aaaagtccag aactctgaaa caccttttca aaagtttttc aagccatgat 150900
    gtttgcaagt taaatgctct gttatgtaag caatataatc agtttttatt aatgtaacat 150960
    tccttagtgt tttggggtat cacacaaaaa agaatatcca tatctggaag caacagcttt 151020
    taaataagag cattgtggtg gtggtggtga tagtggtttt tttttttttt tttgagttgg 151080
    agtctcgctc tgttgcccag gttggagtgc agtggcacga tctcagctcg cttcaacctc 151140
    tgctcccagg ttcaagcaat tcttctgcct cagcctcctg agtagctggg attataggca 151200
    cctgctacca tgcctggctg atttttatta ttttagtaga gacaggtttc accatgttgg 151260
    ccaggctggt cttgaactct taacctcagg tgaatcaccc acctcggcct cccaaagtgc 151320
    tggaattaca ggcatgaacc accatggcca gccaaataag agcattttta atgtaaaatt 151380
    atgcatgaaa tgtacattca attttgtctt tgtttactag gatccatgtt ctcacaagct 151440
    atgaagaaat gggtgcaagg aaatactgat gaggtaaatc ctacctttag gataaaaaga 151500
    tttctgttta taagtgccac cctcatgtaa gtgaggttta aaattttcct tttctttagg 151560
    tcccatgttt aagcagcatg gcacatttat gttctcttac ccagaatgta ccaagaaagg 151620
    gtggtccctt cttaacatct aacaattgcc tggtagtagc agtgaaggta tcttcagtca 151680
    gaggctagga ccactgaagg atatacatgc attcaagttt ccatcagcca gcaggcatca 151740
    gtaatcagtg tgtagatcaa aagctcaaat gtttccttcc ccactggcag ttttacttca 151800
    agtagtggag gcttgctttt ttaatagtta attaagtaca ttgagagatg ggaggtgaaa 151860
    aaaggaaaat gttttatttt gaccatctaa tatgaaagta gttcggtgtt aggtatccag 151920
    tagttgacac tggaagacag ggaatgacat gttaatattc atagccagag ggtggcccag 151980
    gttttttcgt acatgggaat gaaattctta tccaaataag tagaaattat gtgcgtaagc 152040
    catttgttaa gagcactgag tatgtgcatc tcgatccatc taatgaataa ccattatcac 152100
    cagtttaaat tattttcttt aggcccagga agagctagct tggaagattg ctaaaatgat 152160
    agtcagtgac attatgcagc aggctcagta tgatcaaccg ttagagaaat ctacaaaggt 152220
    aaggatgact tcgttttgtg taaactaaaa agtattattt tccaggtgta aaaataaaaa 152280
    agaacataag gggtttcttt gcctttgaag gattaactgc tgtggggatt accttcttat 152340
    cataagcaac tagaaaattg acaaactaaa tgaaacaact gtttgcatat attggacaat 152400
    gggcaataca gggaaaccat ggaaaccaaa cagagcccag tagtcttgct gaacgaaaga 152460
    gttaaatatc aaagttcagg ccaggtgcag tggctcacgc ctgtaatccc agcactttgg 152520
    gaggccaagg cgggtgaatc acttgaggtc aggagttcaa gaccagcctg gccaacatgg 152580
    tgaaaccctg tcttagccgg gtgtggtggc aggcacctgt aatcccaact atttgggagg 152640
    ctgaggcagg agaatcgctt gaaccaggga ggcggaggtt gcagtgagcc gagatcacac 152700
    cactgcactc cagcctgggc gacgagcgaa accccatttc aaaaaaaaaa tcaaagttca 152760
    gagagctcaa tttgagtaga agttgtagga taaggtagca gaaaagagga agctgcccag 152820
    aaagaaagcc gtagagatat ttagagagat tcccatggat ccttggccta ggagtgatct 152880
    gtatatgtgt ggggtgaaaa cgcatgtgtc caggtagaga accccccaga aattagtagg 152940
    ctgaatgatt gctggaacat agggctaaga aaagttcatg gccagaagga tctggccaga 153000
    gtagagagac ttagtaatac acaaggcatt gggtagtgtc ttcacagagg ttatgcctta 153060
    ctactgaaga taaattagtc ctagagtaca agcacctgaa ccaagtttca aagcaaattt 153120
    ttaaagggtc aaattaccta acaactgcat gccaaaacaa aggcctaacc ctctttacag 153180
    taacacaaca aaattcagca cttcacagtg taaagttaga atgtctgacg tccaggctgg 153240
    gcgcagtggc tcatgcctgt aatcccagca ctttgggagg ccgaggcagg tagatgacct 153300
    gaggtcagga gttcaagacc agcctggcta acatggtgca accccgtctc tattaaaaat 153360
    acaaaaactt agccaggcat ggtggccggc acctgtgatc ccggctactt gggaggctga 153420
    ggcaggagaa ttgcctgaac ccaggaggtg aaggttgcag tgagccgaga tcgcaccact 153480
    gcactctggt ctgggcaaaa agagcaaaac tcaggctcaa aaaaaaaaaa gaatgtctga 153540
    cgtcaatcac aaattaccaa gcatgacatg aagttgacct ataaccagga gaaaactcaa 153600
    tctatagaaa cagacccaga tgtgagaaag atgatgaatt tagcagacaa agaccatcaa 153660
    gtggctattt taaatattaa aaatatgttc aagtggccag gtgcagtggc tcatgcctgt 153720
    aatcccagca ctttgggagg ccaaggtggg taggagttca agaccagctt ggccaatatg 153780
    gtgaaacccc ttctctacta aaaatacaaa aaaattagct gggcatggtg gcaggtgcct 153840
    atagtcccag ctatatggga ggctgaggca caagaatcac ttgaacccgg gaggtggagg 153900
    ttgaggttgc agtaagccga gattgtgcca cttgtactcc agcctggaca acagagtgag 153960
    actctgtctc aaaaaaaaaa aaaaaaaagt taaagaaaac aagagtataa tgagaaaaat 154020
    gcaaaatagt tttaaaagaa ccaaatggaa tttcttaaaa taaaaaatac cagaaatggg 154080
    ggccgggcgt ggtagctcac gtctataatc ccagcacttt gtgggggctg aggcaggcag 154140
    atcacctgag atcggtagtt caaggccagc ctgaccaaca tggagaaacc tcatctctac 154200
    taaaaataca aaattagctg ggcgtggtgg cgcattgcct gtaatcccag ctacttggga 154260
    ggctgaggca ggagaattgc ttgaacccgg gaggcagagg ttgcggtgag ctgagattgc 154320
    accagtgcac tccagcttgg gccacaagag tgaaactccg tctcaaaaaa aaaacaaaaa 154380
    aaaacagtag actcgaagaa ctagctgagt ttttctttac tttaggcagt aagtgtgacc 154440
    ttttgcaggt gactacttta gttcctcatg tcctcattag tagatcagag aaattcgaca 154500
    ccaaaacccc aaaagaaaaa ccccttctaa tcctcattcc atgattttat gaatgcatga 154560
    agtcctaggc ctgcgaagga atactcattc tctttatcct gtgttgatac ctctctgctt 154620
    caacctccaa ctcgacattt gcctatagga tgtacttgga cattcagcat aaactacctc 154680
    acaccattac tgaattgctt catgtgcaca tgtcccatgc cacaataccg gggaccttgt 154740
    cttccgtgat atttgtccgc agtgctgtga ctacaggagg gagtcagtga atgtctgcat 154800
    gtgtgtcttt accatccctc ttgaatatgc tctagggtta attcctagaa gtagaattac 154860
    tctattgaaa attggcaata tttttcattc taatatctat tgccaacatg ggaaagcaag 154920
    tctggatgcc agtccttgtt atatgcccct tgggtaagtt acgtaacctc tttaagcttc 154980
    tgttcactca tattttaaca aggaaaatta caatatttta cctcacaaaa ttgtagtcag 155040
    cttctggctg tcttaaactc tggtatatag taaacactaa gtgttggtgt ccatccttaa 155100
    tttgtaataa taggtcactt gttagagaaa tgcaccttac cattttcttt tcttttcttt 155160
    tttcagttat gactcaaaac ttgagataaa ggaaatctgc ttgtgaaaaa taagagaact 155220
    tttttccctt ggttggattc ttcaacacag ccaatgaaaa cagcactata tttctgatct 155280
    gtcactgttg tttccaggag agaatgggag acaatcctag acttccacca taatgcagtt 155340
    acctgtaggc ataattgatg cacatgatgt tcacacagtg agagtcttaa agatacaaaa 155400
    tggtattgtt tacattacta gaaaattatt agttttccaa tggcaataac ccatttatga 155460
    gagtgtttta gcctactgga atagacaggg accacatcct ctgggaagca gataagcata 155520
    gaactgatac ttgatgcaca ctcgtagtgg taactcatcc ctaatcagca ttgtaaagca 155580
    ggtgccagag gtggtttgct ttgtccttcc aaagcaggtg agtcagcccc accgagagcc 155640
    aggcagcttt gagtggcagc gtggtgctag cagcttcagc ggaacagggt gagagttaat 155700
    tatgcagtct tcttgacagc ggcattaatt tggaaggaaa ctgacaagtc atgggtcaag 155760
    tttcagtgac ttcctccttc ctctgatggc agtatatagt tttcacattt taattcctcc 155820
    tcctgagatg cactatactt aaaaccattc tctcccctgc taacagaagg gtgtgaatct 155880
    ggtttacttt gagcattagg atttgcccct ttggaattct gcactccagt tacttaactt 155940
    tcccttcaga atacatgtgg aaagaaagaa agaaatagcg atgactccac ttttgcccct 156000
    gtggcacctt gaacaaagca gttcttccca aattatactt tttttttttt taaataaggt 156060
    gagcaggatg actggggaga gagaaacatt tgactttgac tgcctccccc attctttgct 156120
    gtgagctgga aagtgtgcag ttggtcgtct ttcttctcct ttctttagga tagtaagaga 156180
    ctcactcact gcacttctgc tcagttggct tctgcatcgg gatcacacag ccatcagcag 156240
    gactgcccag ttggtgagca cactccattg accacgtggc gccagcgctt cctcaatgca 156300
    catgattgag aggaaagaaa gttctcttag atgttactgc ttttgctcag actttgcaaa 156360
    aaaaaaaata tatatatata tgtataaata tataattatt aatcactttt gtccttgaga 156420
    aagtcttgaa tgaacagaga atttattcca ttgcaatatt tgattgtata gaggcacact 156480
    gtttcatcga cagaagaagc aaaaaggctt tgtgtaagtt tttggtacta tgtaccacct 156540
    ctgttattct tttaaagctg aagtattcat gtacttaaac catattatat ttaattgtgt 156600
    ttgattttaa aatatatata tatgaattct atttaaaatt gtgtcaactt tctgctttca 156660
    gggcatttat ggctcttctg ttgaaatata ttgatctttc caaatatttt catttgcttt 156720
    ctaaaaaccc agaacatgag ccactactgg actttgcctt gtgtttgaag tgtatggcat 156780
    aaacccaagg tttttattag tcatctatgc tgtgattaat tcattttgtt cttttaacaa 156840
    aatatttcca tccacttcac attgcttcaa tctttaacag aaaagcaata taaaggttat 156900
    agaataaaat gtggttttgg gcaactcttg ctgcctctgc atgttttgga ataacaattt 156960
    ctacaagact ctaggctgtt taaactagtg ctttcagtta agataaattc taatcatttc 157020
    tttgtatata cattttgtgc ttctgagcta gagatgccaa gtagttgtaa actgcttata 157080
    aagagaatag cagcaaattt gagactcggc tacttttttc tgccccacct gctttgagac 157140
    acagaagcgg agtgtggccc gaaattatta gccagattta atatttgatc taaagtaggt 157200
    ccttgtactc attttaaagt tggaatttga ttcctccaac attgagcacc caccatgttc 157260
    caggctctgt gcattgtgcc cacaaaataa gattccctgg tggagttttt atgggttcaa 157320
    ataatcagtt gaacaccctt catctttatc atgttgttga cattgacaca aattgtttaa 157380
    aaagaaaaga tattagagag aaagtggtac ctttgtaact tgatgtgtct tcatcattcg 157440
    gtaagatttg atgaaagtaa aaagcaaatg tcagccaaat ccagtgaaca gcaataaaac 157500
    agggagtaac tttttataac tttttctact tggatttcaa cattcagtag agcttttcga 157560
    aatgtaagta gtttacagta ctggaggttt gactagttca gtaggaattt ggaggggaag 157620
    gtcattctga attgtaacaa agtacaaact tctttgctgt tttatttaag tactgagagc 157680
    taagcacctg atgaagtgac tgacctctct ccagtgacag tgtttgggta cctgcctgac 157740
    ttcaggagtg gggtttatgt ttctacacag tgaccttttc tctcgccctc tcctccctct 157800
    tgcccacaca ccagttgatt ggacctgggt tgaactcctg atccagacag gcccaagaca 157860
    gttcttaatg ttaagaattt tggggccggg cacggtggct catgcctgta attgcaacac 157920
    tttgggaggc cgagacaggc ggatcacttg aggtcagggg ttcgaggcca gcctggccaa 157980
    catggtgaaa ccctgtcttt actaaaaata caaaaattag ctgggcatgg tggcgcacgc 158040
    ctgtaatccc agctacgtgg gtggctgaga caggggaatc gcttgaacct ggaggcggag 158100
    gttgtgcaat gagccgagac cgtgtcactg cattccagcc tgggtgacag agggagactc 158160
    tgtctccaaa aataaaaata agaaaaagaa ttttgggcta ggtgcagtgg ctcacgcctg 158220
    taattacagc attttggaag gcccaagatg ggcagatcac ttgaggacag gagttcgaga 158280
    ccagcctgga caacatggtg aaactccatc tctactaaaa agacaaaagt tagccagatg 158340
    tggtgatggg cacctataat cctagctcct cgggaggctg gggcaggaga atcacttgaa 158400
    cccaggaagc agagattgca gtgagccaag atcacatctc tgcactccag cctgggcaac 158460
    agagcaagac tctgtctcaa aaaaaaaaga atttggccag gcgcagtggt tcacgcctgt 158520
    aatcccagca ctttgggagg ccaaggcagg cagatcacga ggtcaggaga tcgagattgt 158580
    cctggctaac atggtgaaac cctgtctcta ctaaaaatac aaaacattag ccgggtgtgg 158640
    tggtgggcac ctgtagtccc agctactagg gaggctgagg cagaggaagg atgtgaaccc 158700
    aggaggcgga gcttgcagta agccaagatc gtgccactgc actacagtct gggcgacaga 158760
    gtgagactcc gtctcaaaaa aaaaaagaat tttggccggg tgcggtggca catgcctgta 158820
    gtcccagcac tttgggagac caaagtgggc ggattacctg aggtcaggag ttcaagacca 158880
    gtccggccaa tatggcgaaa ccctgtctct tactaaaaaa aatacaaaaa ttagccaggt 158940
    gtggtggcgg gcacctgggg aggctgaggc agggagaaat gcttgaaccg gggaggcaga 159000
    ggttgcagta agccaagatc gtgccactgc actccagagc aagactcttt ctcaaaaaaa 159060
    aaaaaaaaag aattttgcat ggggaaggag agatactgtt caccatctgg aatggtgctt 159120
    ggatgtggca cttacaaaat caggagccag cactgcatgg acaaacagaa gcatgtgggc 159180
    ctgagatagc aggtaccttg ataaccctga agacatcctt ggtttctgca tctattcctg 159240
    catccttgca ttggactaca ttaatctgtc agttatcctt ataatgattt ttgatttttt 159300
    ttttttgaga tggagtttcg ctcttgttgc ccaggctgga gtgcaatggc acgatctcgg 159360
    ctcaccacaa cctccacctc ccaggttcaa gtgattctgc tgcctcagcc tcctgagtaa 159420
    ctgggattac aggcatgcgc caccacacct ggctaatttt gtatttttag tagagacggg 159480
    gtttctccat gttggtcagg ctggtctcga actcccaacc tcaggtgatc accctgtctc 159540
    ggcctcccaa agtgctggga ttacaggcgt aagccatggt acccggtctg ttttttgatt 159600
    ttttgaaacc agtctgaagt gagttttttt aattacgtga aaggagtttg gctaaaatac 159660
    tgccatactg ccctaatgcc taatgattat gtattctcag catgtctgca aagtactgct 159720
    gatttctgga gaataatttt tctttagtaa acttcactta agtcgtcatg tgtattctct 159780
    caaaatggta tcctaaccta atggagctaa aagacacccc ttgtttttat aacaagcagt 159840
    tactgaggcc caggaagggg agaagtccct ggcttgtgag atgatcacca ttagaactca 159900
    ggcctgggcc agtgcctttt catgcttctc agatccttcc aaagaataat gaagattata 159960
    accgctttta gcaattgtaa taaacccaga aatagaaagc tttttggtta gagtactggt 160020
    agaagtttgg cgggagagat aatttttaca aaatttgtaa atacctgcca attctatata 160080
    ctaggcaagg tctctggcct tgtaaaaccc ctcaaggtta caactttggt ggcccacact 160140
    aatagttacc cactgaggcc ctctccgggt gaacattgag cactagagga agcccctctg 160200
    cttgggcagg actgggcgtg gtgcagagta ggagcggtga tactgtggat tctgggcagg 160260
    tggagatggc cagtgatgtc caataaagga cactggaggg agcagtgtga gtaaaggccc 160320
    tgagggcatt catgttcagg gagggttgct gcccactggc ttgcttggca cacaggagag 160380
    tgggtattcc tgccttagta actttatgta aacaagtatt tcctcagtct gttcctctca 160440
    aactgcctgc tctggcacat tcagaatgtc acagaactca cctggatgca ttcagcccct 160500
    tgcctaaagg tgacagtgca tctccttccc caccccaccc ctcataccac tgaagcacct 160560
    gtcagactgg cccagtctgt gggcaaggag cctagagagg gcttagtttc agcttgaaag 160620
    gagctgggat ttaccaagaa gcaaatgaga gacgaggatt gcaacaactg tgccatttcc 160680
    ccagcttcag ctgactcctg tatattgact gtgccttcag actcatccgt aagtgacccc 160740
    aggctggcct ctcccacatc acagtaagaa ttccacacac catacaactt ggaaagaggc 160800
    tccagctgaa ggaagcccca cacttctttc aagtttttct tagtcttctc ttcttggcaa 160860
    agagtacctt ttgtttcttc taattatgta actattggtt tagtaaatat tcacccattc 160920
    agtcaccctg taagtggcag gcactgttta cagggacaca ggaaggaata aaaacttgca 160980
    ggcaccttgg agcttgcatt ctattgaaga ggtaatggaa gttgggatag cagctaaact 161040
    atgctggtat tggccaggcg cagtggctca cacctgtaat cccagcactt tggaggccaa 161100
    ggtgggcaga tcatgaagtc aggagatcga gaccatcctg gctaacatgg tgaaaccccg 161160
    tctctactaa aagtaaaaaa aaaaattagc caggtgtggt ggcgggcgcc tgtagtccca 161220
    gctacttggg aggctgaggc aggagaatgg tgtgaaccca ggaggcgaag attgcagtga 161280
    gccgagatgg caccactgca ctccagcctg ggtgacagag cgagactctg tctcagaaaa 161340
    aaaaaatatg ctggtagttt tgattcaaga tggcctttgg agcccatgat ttaggtctcg 161400
    tacccaccaa ggtctactgg aaaacatcag gctctcctgc tatagaccca tagggagagc 161460
    tgcagccgag agggggagct gaagagaagt gccccttctg tgtcctgtca gcctcatcct 161520
    tccgcaagga ccagttgctg tgccactcca ttcacttgct gcaagactgg aggtttttcc 161580
    tcaggtgttg agcacctggt ttacaagatg tcagcatctt gatgcctgag accatcaagg 161640
    caagtctctg aacagggctt accttagagt aaggcttaga agaggccgta aagtcagtct 161700
    cagctccgtg gctctgcaga gctttgggac atgtgaattc ttaaaaacaa gactattgta 161760
    cagttactat atgcatgcag tataaaatta taaccttgga aaatcctagc tagctgttga 161820
    gctaattcca taaagtaatc agctcctgag ttctgcagtg gtaataataa tcagcataat 161880
    gagtaaacac tgtgtgtgcc aggcagcgtc tcatttgatc cttgtgataa tcttgtaagt 161940
    actgattttc tcccttcttt aaacaaagtt tttttttttt ttttagagag ggtctcacta 162000
    tgttgcccag gctagtcttg aattc 162025
    <210> SEQ ID NO 36
    <211> LENGTH: 162025
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <220> FEATURE:
    <221> NAME/KEY: mutation
    <222> LOCATION: 156,277
    <223> OTHER INFORMATION: Nucleotide Base Change: T to C
    <400> SEQUENCE: 36
    gaattcctat ttcaaaagaa acaaatgggc caagtatggt ggctcatacc tgtaatccca 60
    gcactttggg aggccgaggt gagtgggtca cttgaggtca ggagttccag gccagtctgg 120
    ccaacatggt gaaacactgt ctctactaaa aatacaaaaa ttagccgggc gtggtggcgg 180
    gcacctgtaa tcccagctac tcaggaggct gaggcaggag aattgcttga acctgggaga 240
    tggaggttgc agtgagccga gatcgcgcca ctgctctcca gcctgggtgg cagagtgaga 300
    ctctgtctca aaaagaaaca aagaaataaa tgaaacaatt ttgttcacat atatttcaca 360
    aatttgaaat gttaaaggta ttatggtcac tgatatcctg tttcattctt tatataatca 420
    ttaagtttga aatgtatact tgcactacta acacagtagt taatcttagt cctacaagtt 480
    actgctttta cacaatatat tttcgtaata tgtatgcact ggtgtttatg tacgtgttta 540
    tgtttatatc tgttaaaatt agcagtttcc atctttttct attttgtacc atcacatcag 600
    ttcagaagga ttgacagagc aaaatgattt gatgaagtat aaaagtcaca tggtgagtgg 660
    cataaataca actctgaaca attaggaggc tcactattga ctggaactaa actgcaagcc 720
    agaaagacac atatcctata tgtcaagaga tgtaccaccc aggcagttaa agaagggaag 780
    tacacataga aagcacaatg gtgaataatt aaaaaattgg aatttatcag acactggatt 840
    catttgctcc taaagtcaga gtcctctatt gtttttttgt ttttgtgggt ttctttttaa 900
    atttttttat tttttgtaga gtcggagtct cactgtgtta cccgggctgg tctagaactc 960
    ctggcctcaa acaaacctcc tgcctcagct tcccaaagca ttgggattac agacatgagc 1020
    cactgagccc agcccagacg ctttagcatt tatgaagctt ctgaaatagt tgtagaaacc 1080
    gcataagctt tccatgtcac tttcaaagtt tgatggtctc tttagtaaac caaccaagtt 1140
    attcctcaag ggcaaaataa catttctcag tgcaaaactg atgcacttca ttaccaaaag 1200
    gaaaagacca caactataga ggcgtcattg aaagctgcac tcttcagagg ccaaaaaaaa 1260
    aggtacaaac acatactaat ggaacattct ttagaagagc cccaaagtta atgataaaca 1320
    ttttcatcaa agagaaaaga gaacaaggtg ttagcaaatt cctctatcaa ataacactaa 1380
    acatcaagga acatcaatgg catgccatgt ggaagaggaa gtgctagctc atgtacaaac 1440
    cagtagataa tttcaacttg ctgccgaatg aaacctcttt gcaaggtatg aatcagcact 1500
    tctcatgttt gttttgcttt gttttgtttt gtttttagag acaggccctt gctctgtcac 1560
    acaggctgga gtgcagtggc acgatcagag ctcactgcaa cctgaaactc ctgggctcaa 1620
    gggatcctcc tgccttagcc tcccaagtag ctgggactac aggcccacca tgcccagcta 1680
    attttttaaa ttttctatag agatgggatc tcactagcac ctttcatgtt tgatgttcat 1740
    atacaacgac caaggtacaa tgtggaaaag ggtctcaggg atctaaagtg aaggaggacc 1800
    agaaagaaaa ggggttgcta catagagtag aagaagttgc acttcatgcc agtctacaac 1860
    actgctgttt tcctcagagc agagttgatg atctaaatca ggggtcccca acccccagtt 1920
    catagcctgt taggaaccgg gccacacagc aggaggtgag caataggcaa gcgagcatta 1980
    ccacctgggc ttcacctccc gtcagatcag tgatgtcatt agattctcat aggaccatga 2040
    accctattgt gaactgagca tgcaagggat gtaggttttc cgctctttat gagactctaa 2100
    tgccggaaga tctgtcactg tcttccatca ccctgagatg ggaacatcta gttgcaggaa 2160
    aacaacctca gggctcccat tgattctata ttacagtgag ttgtatcatt atttcattct 2220
    atattacaat gtaataataa tagaaataaa ggcacaatag gccaggcgtg gtggctcaca 2280
    cctgtaatcc cagcacttcg ggaggccaag gcaggcggat cacgaggtca ggagatcgag 2340
    accatcctgg ctaaaacggt gaaaccccgt ctactaaaaa ttcaaaaaaa aattagccgg 2400
    gtgtggtggt gggcacctgt agtcccagct actcgagagg ctgaggcagg agaatggtgt 2460
    gaacctggga ggcagagctt gaggtaagcc gagatcacgc cactgcactc cagcctgggc 2520
    gacagagcga tactctgtct caaaaaaaaa aaaaaaaaaa aaagaaataa agtgaacaat 2580
    aaatgtaatg tggctgaatc attccaaaac aatcccccca ccccagttca cggaaaaatt 2640
    ctcccacaaa accagtccct ggtgccaaaa aggttgggga ccgctaatct aaataatcta 2700
    atcttcattc aatgctaaaa aatgaataaa ctttttttta aatacacggt ctcactttgt 2760
    tgcccaggct ggagtacggt ggcatgatca cagctcactg tagcctcaat cacccaggcc 2820
    ccagcgatcc tcccacctaa acttcctgag tagctgggac tacaggcacg caccaccatg 2880
    cccagctaat ttttaaattt tttatagaga tgggggtctc accatgttgc ccagactggt 2940
    ctcaaaccct gggctcaagt gatcctccct caaactcctg gactcaagtg atcctccttc 3000
    cttggcctcc caaagtgctg ggattacaag catgagccac tgtacccagc tggataaaca 3060
    ttttaagtcg cactacagtc atggacaatc aggcttttca acatgcagta tggacagtga 3120
    gtcccagggt ctgcttttcc atactgaaat acatgtgata ctaaggagaa aggtgctcgc 3180
    aaggatattt aaaatgaaga atatttaaaa tgaggaaaaa actgtttctt catgactttg 3240
    ataaggctga taaagaccat ttctgtgatc tcaggtgatt cactcaagta gtatatttca 3300
    gtaatcatta tctggaacag cctgaatctt aaccaaaata ccatgatttt ttaatgctgt 3360
    tatgatacct tgatgatatg accaaactgc aatgtaggca gctaaatctc cacgagtttg 3420
    acttccccga gagttgacag ttttcttcac aaattaaaga aatatatttt ttgatacatg 3480
    attggcatat ttaaaaacta cactgaaatg ctgcaaaatg atataaagaa acattttcca 3540
    gaatcaaatg caatcaaaga gtggattagg aatctactca ccattatcaa ctaaatagaa 3600
    acacttggac tgggtgtggt ggctcacatc tgtaatctca gcactttggg aggccaaggc 3660
    aggtggattg cttgaggcca ggagctcaag accagcctga gcaacatagc aaaactctgt 3720
    ctctacaaaa aaaaaaaaaa attaaccagg catggtggca gatgcttgta atcccagcta 3780
    ctctggaagc tgaagtagga ggactgcttg agcccaggag atcaagactg cagtgagccg 3840
    tggtcatgct gcgccacagc ctgagtgaca gagagagacc ctgtctcaaa aacaaaaaca 3900
    aacaaaaaac acttaacctt cctgtttttt gctgttgttg ttgttgtttg tttgttttga 3960
    gatggagtct cactctgttg cccaggctgg agtgcagtgg cgtgatcttg gctcactgca 4020
    agctctgcct cccgggttca cgccattctc ctgcctcagc ctcccgagta gctgggacta 4080
    taggcgcccg ccaccacgcc cggctacttt tttgcatttt tagtagagat ggggtttcac 4140
    cgtgttagcc aggatggtct tgatctcctg acctcgtgat ccacctgcct cggcctccca 4200
    aagtgctggg attacaggca tgagccaccg cacccggcca acctttctgt tttttagttt 4260
    gatatgcttg ttaactcagc agctgaaaga atgctgaaag tggccttcag taaaaaaatt 4320
    tcactagaat ctctacatcc atatttaatc tgaatgcata tccagattga tcagttagag 4380
    caaaaacact catcatcatt cctgatgacc tctaattctg gtttcggctt tctatttcaa 4440
    tggaaacaga ataaggaaag aaatggaagg gctctggaaa tttgtcctgg gctatagata 4500
    ctatcaaaga tcaccaacaa taagatctct cctataaata taaaacaagt ataattaatt 4560
    ttttaattat ttttttctct tcagaggatt ttatttcaag ataaaacata acttctaccc 4620
    atactattga ttccaaaggt tagaaaaagt gtttttcctc atcttatcct tcaaagaggt 4680
    cacagcaatg caaacatcta taaaatgcct ctgcataatt gtcagaagct atagtccaga 4740
    aatcattgaa aatgcttttc cattttaagc ttaggtgagg tgtcttagga aacctctatg 4800
    acaacttact ctatttattg ggaggtaaac tcccagactc tcccagggtc tcctgtattg 4860
    atctcatttt ttaggcttcc taatcccttg aagcacaatc gaaaaagccc tggatctctt 4920
    ttctgcacat atcatcgcgg aattcattcg gcttccagca agctgacact ccatgataca 4980
    agcggcctcg cccttctccg gacgccagtc cttgctgcgg ttagctagga tgaggggttt 5040
    gctgggcttc agtgcaggct tctgcgggtt cccaagccgc accaggtggc ctcacaggct 5100
    ggatgtcacc attgcacact gagctcctgg caggctgtac caatttttta attatttaat 5160
    atttattttt aaaattatgg tgaatatttt ggtattctgc tctaaaatag gcccataaat 5220
    gcacagcaga tatctcttgg aacccacagc tttccactgg aagaactaag tatttttctt 5280
    ttaaagatgc tactaagtct ctgaaaagtc cagatcctct acctctttcc atcccaaact 5340
    aagacttgga atttatgaga gatctagcta acagaaatcc cagacacatc attggttctt 5400
    cccagagtgc agtcctccta aagaggctca gccctaagca ggcccctgca ccaggagggt 5460
    gggtctgaga cccacatagc acttcccaag gtgcatgctc cagagaggca ctgaaacagc 5520
    tgagcacaag cctgcaagcc tggagaactc tcacagtcag aacggagggg gcccagtggg 5580
    actaacataa agagaaaagg gaacacagag aaatggatgg caccaacaac cagcaaagcc 5640
    ttcatggcca atgaaagcat cagtgacggg gccagaaccc tcatccccaa agactcttca 5700
    ctgcctttag tgaaaaacaa tggctagaga gtgaagttat gatcatgtat agagaggtaa 5760
    agttacattt ttatattctg actctgctaa tgtgaaattc cctatctgct agactaaaag 5820
    tttcagacac cctgttcaaa tatcccatta gttgctagag acttaaaatg aacagaacgc 5880
    acattgtcag gatgactatt accaaaaaat caaaagacag caagtattgg tgaggatgta 5940
    gagaaactgg aacttttgtg cactgtttat gagaatgtaa aatggagcag ctgctgtgga 6000
    aaagagtatg caggttcctc aaagagtaaa accaagatgt ggaaacaact aaatgcccat 6060
    cagtggatga aggggtagac aatatgtggt atatacatac catggagtac tattcagcct 6120
    ctaaaaaaaa aaaaggaaat tctataacat gcaacagcat ggatgaatct tgaggacatt 6180
    ttgctaatga aataaggcag tcatagaaag acaaatactg cacgactcca cttatatgag 6240
    ataccaaaaa tagacaaatt catagaatca aagagtacaa tggaggttac ctggagctgc 6300
    agggcgggaa acgaggagtt actaatcaac gaacataacg ttgcagttaa gtaagatgaa 6360
    taagctctca agatcagctg tacaacactg tacctagagt caacaataat gtattgtaca 6420
    cttaaaaatt tgttaagggt agattaacaa atgtagtaga tccacaaatg tggttaagtg 6480
    ttcttaccac agtaaaataa aaaaagaata tcaagcccag gagttcgaga ctagcctggg 6540
    taacatggtg aaaccctgtc tctacagaaa atacaaaaat tagccagctg tggaggtgca 6600
    ctcctaggga ggctgaggtg ggaggcttgc ttgagcccag gaggtcaagg ctgcagtgag 6660
    ccatgattgc accactgtac tccagcccag atgacagagc aagacaccac cccccccaaa 6720
    aaaagaaaaa gaatatcaaa cattttaaaa gatcagatac gcaagaacaa caacaaaaaa 6780
    gagatgaaca gagcatcgac cctcatctag tgggattctt ggtctaactg aaaaacagac 6840
    attgagagac aaacaatgac agtgatgtga tcacagcaat tacacaggta tcccctgggg 6900
    actgcagaag aaaggaggaa tgcctaactt tcagaaaata gagaaagcgt caaacagttg 6960
    gtgaaagcct tccaaaacta gagagaactg cacacaccaa atcacagaaa gaagaaaagc 7020
    cgtgggagat tctgggaccc accggctatt tttgatggct gaacaccctg ctgcaggaga 7080
    gacaggagct ggaaagcatg gtgggatgaa acctcaaaca gctttgcctg cattgcttaa 7140
    gatgactggg cttgattaac tctagtcaat ggggacaatt caatcaaaga agaaagatgc 7200
    tcaaattcac attttagaat gattttttat ggcagtatgg ggaatagatt aaaagagagt 7260
    gaagctggag gcaagaaact tgttaagagg caactgaaac agtctagatg ataaataata 7320
    aactgacaga gtgactagaa aaatcagaac aggctgaatc aacagatacc tagatgaaaa 7380
    taacaggact tgatcaccag ttgtatcttg gagaggaagg agttgtttcc ttgctttccc 7440
    tacgactggg aatacggaag gtttgccgtg tgtattggtt atatactggt gtgtagccaa 7500
    tcactgacaa ccatttagca gcttaaaaca caaaggctta tctcccagtt tctgtgggcc 7560
    aggaatctaa gataggctta gctggctggt tctggctcag agtttctcaa gaggttgcaa 7620
    tcaagatgtc agctggggtt gcatcatctg aaggctcaac tggggccgga gggtccactt 7680
    ccaaggagtt cactcacctg cctgacaagg cagtgctggt tgttggcagg agatctcaat 7740
    tcattgccaa gtgagcctct ctatagcatt gctggaacat cctccccatc tggcagttgg 7800
    cttctctcag catgagtgat ctgagagaga gagcaaggag gaagccacag tgttcttcct 7860
    actcctactc ctaacactat ggacctactc ctaacactct cacttctgcc ttattccatt 7920
    agttagaaag ggaactaagc tccacctctt gaaataagaa gtgtcaaaga atttgtggat 7980
    atatttaaaa atcatcacac tgtggaagtg gatagggggt tcaattaatg ctgaacttga 8040
    aatgcctgag acattcaaat gtccaacagg caatgaacat acccatagat ggtcatgact 8100
    ttagcaagaa tagaggaaga tcacagaatt aaggaggaat tgaaaggtaa aagaagtgga 8160
    gtcagattcc ccctgaaaag tgagccatga aaggaacttt aactattgag ttagaggtca 8220
    gagtaggaaa tttcggtgga attctttttt aaagaaagga accatataag catgttttga 8280
    ggtagaggga gaataaatca gtagacaggg agaggtaaaa aacataaatg ataggggata 8340
    gttgacaaag gtcttggcag aatcccttac ccattgactt ggggccaaga gagggacact 8400
    tctttgtttg agggataagg aaaataagaa agaatgggtg ctatttagtg tggtcctgtc 8460
    tctagggcaa acgcataggt aacaaactgt gtgtgttagg aatatagatg tgacctcaca 8520
    ttgagattct cacctcaaat ccattttgtt gttacctgta ccttcctacc ttctcttttt 8580
    gctacatgca gactgctgtt ttgtcttcct ggcctgttcc aggtttcagc attctggcat 8640
    atctgctacc ctgttcccaa acctctctag agtccatgct ccttccttgg atagtgtttg 8700
    attgggccac gtatctaaga agtgatgcct tcagttaggc ctgagaacct cctctatgga 8760
    aatctccatc agtgaccctg acagacttgg tatcttggag atgtcactgc tcccagcctg 8820
    tggtctagga gaatctcagc ctgggcctct agtagtatgg ataaggcgtt aaggtatctt 8880
    tgaaccagag tctgtcatat tcctcaatgt gggacagata aaacagtggt agtgctggtg 8940
    tttctgagct agaactctgg tttttggtct agattctttg atgtatgacc tttcagaggt 9000
    attaaaattt gttctaatac aatgttcaat acaaatgtag ttccttttct gttaggacct 9060
    caacaaaaca tgaccaactg tagatgaaca ttaaactatg acaattcatg gaaatgaata 9120
    cagtaatacc tgcggttccc ccattttagc agtcactatg gtgacatttg gcacaaatgg 9180
    ctatttaagg gtgcttttgt taaaacctac catcttacta ggcacatgat attgaaacta 9240
    atgaaataat ggagaaactt cttaaaaact tttaatgaat aaagtgatga agtgataata 9300
    ttttagctgc tatttataaa gtgactatta caggtcaaac attcttctag ggtttttttg 9360
    ttgaagttgt cacatttaat ccttaataac ccactatgag tcaggtattc ttctctcccc 9420
    tttggacagt tggggaaatg ggggtcagag aggttaggta atttgctcag ggccacacaa 9480
    cctgcatgta gaaaatctga gatttgtaca ggaacgtatc aaactctgaa gtccatgctt 9540
    ctattttccc atgctgcctt tctaataaaa ggtaactaat gctactggat gctgccccca 9600
    aagtgagtca ctttcacccc accctacttg attttctcca taaaactaat cacatcctga 9660
    caacttattt attgctgatc tcccccacta gattataaac tcaataaaag caagatcctt 9720
    gtctgctgaa tatcagtacc taaaacgctg tctagcacag agcaagtaat taatatttgt 9780
    tgaatgaaca aataaaggaa aaaaattcaa aggaagaaaa agccctaaaa cagatgttta 9840
    cctaaacata cattttaaaa gaaagcatat aacaaattca ggacagaatt taaatttgat 9900
    tttttaaaga aataaccaag tgctagctgg gcacagtggc tcacacctgt aatcctagca 9960
    ctctgggagg ccgaggcagg cagatcactt gaggtcaaga gttcaagacc agcctggcca 10020
    acatggtgaa acctgtctct actaaaaata cagaaattat ccaggcatgg tggcaggtcc 10080
    ctgtaacccc agctactcag gaggctgagt caggagaatt gcttgaaccc aggaggcaga 10140
    ggttgcagtg ggccaagatt gcaccactgc actccagcct gagtaacaaa gcaagactct 10200
    gtctgaagga gaaggaaaga aagaaggaaa gaaggaaaga aggaaagaag gaaagaagga 10260
    aagaaagaaa gaaagaaaga aagaaagaaa gaaagaaaga aagaaagaaa gaaagaaaga 10320
    aagaaagaaa aagaaagaaa gaaagaaaga accaagtgct tatttgggac ctactatgct 10380
    atgtttttcc atgcacgcta ttttcagtaa agcagttagc aaacttgcaa gatcataaca 10440
    acaaatatat gcttctataa ctctaaaatt gtgctttaag aagttcctct ttaccagctc 10500
    atgtatgcat tagttttcta agagttacta gtaacttttt ccctggagaa tatccacagc 10560
    cagtttattt aaccaaagga ggatgcttac taacatgaag ttatcaaatg tgagcctaag 10620
    ttgggccagt tcatgttaat atactccaga acaaaaacca tcctactgtc ctctgacaat 10680
    tttacctgaa aattcatttt ccacattacc aaggagccag ggtaggagaa tatagaaaga 10740
    ccacccaaga atccttactt ctttcagcaa aatcaattca aagtaggtaa ctaaacacat 10800
    gccctaacaa tgaatagcag attgtgctca gaagaatgat ctacaacatc ttactgtgaa 10860
    ggaactactg aaatattcca ataagacttc tctccaaaat gattttattg aatttgcatt 10920
    ttaaaaaata ttttaagcct aaattttaaa aggtttgata ttggtacatg aatagacaaa 10980
    cagacatgga ctagaccaag aattaggttc aaacatatac aggaatttaa tatacgataa 11040
    atctagtatt ccaaaggaac caacaaatgg tgttcagaca gcaggatagg catcaggaaa 11100
    aacacagttg ggcaccctac cttactccta acaccaggag taactgaagg agcaccaaat 11160
    atttatttat tttaattata gttttaagtt ctagggtacg tgtgcacaac atgcaggttt 11220
    attacatagg tatacatgtg ccatgttggt gaggagcacc aaatatttaa aagaaaaaaa 11280
    ttggccaggg gcggtggctc acacctgtaa tcccagcact ttgggaggcc aaggtgggca 11340
    gatcacctga ggtcgggagt tcgagaccag cctgagcaac atggagaaac cccatctcta 11400
    ctaaaaatac aaaattagcc aggcatggtg gcacatgcct gtaatcccag ctacttggga 11460
    ggctgaggca ggagaatagc tttaatctgg gaggcacagg ttgcggtgag ctgagatatt 11520
    gcactccagc ctgggcaaca agagcaaaac ttcaactcaa aaaaattaat aaataaataa 11580
    aaataaagaa agaaaagaaa aaaatgaaaa tagtataatt agcagaagaa aacaccgtag 11640
    aatcctcgga ctcttaggat ggggaatgcc tataatataa aaaccctgaa gttataaaag 11700
    agaaaatcac ctacatacaa accaaatctt tctacatgcc taaaacatag cacaaacaca 11760
    gctaaataat catagctgaa tgaactggga aaacaaaact tgactcatat ccagacagag 11820
    ttaattttcc tacacataaa gagtacctat ataaacccaa caaaaaaacc accactaacc 11880
    caaaataaaa atgtgacagg taatgaacag gtagttcaca gagaatacaa atggctcttc 11940
    ggcacataag atgctcagac tgacttttac ttatttattt tttgagagac agggtctcac 12000
    gatgttgccc aggttaggct caaactcctg ggctcaaatg atagtaccag gactacaggt 12060
    gtgccccacc gcacctggct cctcaaccac ctgtattaac aggaaatgca aaataaaact 12120
    ttcaaatcta ttttacctat tagaatggca aaaatttgaa aaacttcaaa catcatcatg 12180
    ttggtgagaa tgtgaggaga ctggcactct cattttttgc tgatagcata tatatactga 12240
    tggcttctat ggaaagcaat ctggcagcgt ctatcaaatg tacaagtgca tatatccttt 12300
    gacaaagcaa ttccactcta ggaatgtgtt ctatatggtt gtgcttcctg gggctgggaa 12360
    ctgggagcta agggacaggg gcagaagata atcttctttt ccctccttcc ccgttaaaca 12420
    tgttgaattt tatatactgt aatatattat ttttcacaaa agataatttt taagcgatat 12480
    gtctgggaat tttttttttt cttttctgag acagggtctc actctgtcat ccaggctgga 12540
    atgccatggt atgatctcag ctgactgcag cctcgacctc ctgggttcaa gcaatcctcc 12600
    cacctcagcc tcctgagtag ctgggactac aggcacgtgc catcatgcta atttttgtat 12660
    atacagggtc tcactatgtt gcccaggcta atgtcaaact cctaggctca agcaatccac 12720
    ccacctcagg ctccaaagtg ctgggattac aggcgtgagc caccgcgcct ggccctggga 12780
    attcttacaa aagaaaaaat atctactctc cccttctatt aaagtcaaaa cagagaagga 12840
    aattcaacct ataatgaaag tagagaaggg cctcaaccct gagcaacaaa cacaaaggct 12900
    atttctgaga caggaatttg ctgaacaaaa tcgagggaag atgacaagaa tcaagactca 12960
    cttctcggct gggcgcagtg gctcacacct gtaatcccag cactttggga ggccgaggcg 13020
    gacagatcac gaggtcagga gattgagacc atactggcta acacagtgaa acccagtctc 13080
    tactaaaaat acaaaaaatt agccgggcgt ggtggcaggt gcctgtagtc ccagctactt 13140
    gggaagctga ggcaggagaa tggcgtgaac ccaggaagcg gagcttgcag tgagccgaga 13200
    tcacgccact gcactccagc ctgggtgaca gagcaagact ctgtctcaaa aaaaaaaaaa 13260
    aagactcatt tctctagatc ttgagccgta ttcaaattta tctcagctta gtgagaggtt 13320
    aaagcaagga atatccttcc ctgtgggccc tgctccttac tgaaggaagg taacggatga 13380
    gtcaaggaca ccaatggaga aaagcactaa caccattatc tgatgaacat tacgtgaaga 13440
    agggtaagaa gtgaagtgga attgctgaag aagtcagtga aagcggacat tcatttgggg 13500
    aaatggaata taggaaatcc ataaaagtga ttaaaaagat gttagaggct gaggcggggg 13560
    gaccacaggg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc catctctact 13620
    aaaaatacaa aaaattagcc aggcgtggtg gcaggcacct gtagtcccaa ctactcggga 13680
    gactgaggca ggagaatggc atgaacctgg gagacggagc ttgcagtgag ccgagatcac 13740
    gccactgcac tccagcctgg gtgacagagt gagactccat ctcaaaaaaa aaagttagat 13800
    acgagagata aagatccaac agacacacaa ctgctaattc tgaacagaac aaaacaaatg 13860
    gcacaggaaa agaaaattta agatataaca ccggaaaact ttcctgaaat tgagtaactg 13920
    aatctatagc ttgaaagggt ttagcatatg ccaagaaaaa tcagtagagt ccaaccagca 13980
    caagacacat ctagcaaggc tggtgattct accaacacag agaaagaagt gggtgaccca 14040
    taatgcggaa aaaggcagac catctgcagt cttctccaga acactggagt ctgaagacaa 14100
    aagaatgctg cctactgagc cagaagggag agaaagtgac ccaacacatc tttaccaagt 14160
    tagaatgtca cgcattattt aaaggctgca aaagccatga aagacatgaa agaacacaag 14220
    catttacaac atgaaagaac acaagcattc tcatactcaa gaatccttaa gaaaaatgta 14280
    gtcctaatcc agcccactga aagttaaatg tacttaatgt gctcattaat gggaacttca 14340
    tagcttcaaa tcagtctggt cccatctacc aacatctctc gcccggcttt cctgcaatag 14400
    tcagcacctt tccctcctcc cagtcttgtc ccctggagtc tgctctcagc atagcagagt 14460
    gaccacatca acacccaagt cagagccctc cagtgcgcac tggtctacaa agcccttccc 14520
    accccccacc ccacgtgccc tccggatcct tgtgacgtgt ctcctgcata ccctagcagc 14580
    cctggcctcc tcactgcccc tcctgtacat caggaaggcg actccttgag tcttggctct 14640
    ggccgcctcc tccacctgca gtgagttaac tcccttacct actctaggtc attgctcaaa 14700
    tgtcagcatc tcaatggggc cctccctgac taccctattt aaattctaca tactcccctt 14760
    gaccccatgg acctcactca ccctattcca cttttattct tacaatttag cacttgttct 14820
    cttctaacgt attctaagac ttactcattt attacattgt ttgccacccc ctctagtaca 14880
    taaactccag aggggcaggg atttctgtct atttattcat ttctttatcc ctaggacata 14940
    gaacagggca tagttcagag tattcaatgt tatcaatgaa tgaactagca gtagtaccag 15000
    ttccagttag gcacagaatt aaatctaaat agaattaaat ctcatggtct gggttaacta 15060
    tggatagaaa attagatata attttaagaa gcctagaaag aaaaaattaa taatgtaaaa 15120
    ataatattaa tttgataata ataacaaaaa ctctgccagg cactgtggct caaatctgca 15180
    atcccagcta ctcaggaggc tgaggtggaa ggatcacttg agaccagagt tcaagactca 15240
    gcctaggcaa cacggcaaga aactgtctct aaaaaaatta aaacttaaat ttttaaaaaa 15300
    gaattctcaa agcgtcacaa aaactggaga ttaaggtaca ggaagtgtga agtaatatta 15360
    ctatgctaat ggtttttttt ttttttagaa aggtataacc aaaagatttc tttctcaagt 15420
    cgataaactg agaaagataa gcatatcttc caattaacag agggggagga aaagccagat 15480
    acaacaaaat aagatataaa ttagtttcca gttgaaaaca agagtaggag ttattttgca 15540
    tcacctcacc tgtgacctcc cccagcccaa aaaacactac tgataaacag ggtagaaaag 15600
    catcatctca gataaagcag gaaaaactgc cacagtctca aaccacaaac tataagcaca 15660
    cacctggcca accctgccaa gtctgggctc agtaggagga acgtgctgag agctaggatg 15720
    taccaactta gacattctgt gggatacaga tgtccctgga agggtcacac catctcaaag 15780
    gcacctgtaa tgcccactga ttacagccac catatgtgag agagaaactc agggcactta 15840
    gagagtataa caagaacctt atgtcatctg agatgaggaa tcctcagccc tgcaaattaa 15900
    ccaactcttt agaacaactg gcaaaacata aatatccaca acttttgttt cagtaattcc 15960
    actcttagat atcaatccaa agtacatgag acagcagata cacacacaaa atggtattta 16020
    ctgcagcatt gtttataata gcaaaaaaca agaaataatc catatgtctc aataggatac 16080
    tgggtacatg agggtatgta cccatcattc aaccatcaaa aagagtgata tggatgtcca 16140
    cagatggaca taaaaagctg tgtgttacgt gaaaacaaac tcaagcagca gcaggatggg 16200
    cttatgatag tcagtatgag ctaatttctg gaaaaaaaaa tctagtgtgt gcacagaaaa 16260
    catctgaaag aacagaaaca aaactatcag cagaatattg agatgtttta ctaagttgta 16320
    tatctatact gcttgtaatt tttaccccaa gcaagaatta ctttttggaa aaagaaaatt 16380
    caggaaataa agcatttctt taaacttcat gtttaaacaa atggtgatgg aataaaagag 16440
    ttcttattca tcataaacac acacagcaca catgcacgca tgtgcgtgag cacacccttt 16500
    acttgataaa taccatgttg aatattttag tctttccttt taggttctat cccttcactc 16560
    aaaatgcggt tataaataaa tgtacttttc atgtgccttc tgcctaaacc cactttaata 16620
    taactttaca gtcccattat cattatagtc tcaaagctag actcagcctg aaactaccct 16680
    ttcatttgga acccttatta aaatgccaca tacagctcct tcaaataaaa acaaacccta 16740
    ggacctgaca ctaggcttcc tttgttgcta ctcataatgg ccaagttctg tgcttataat 16800
    acatcttctt tcattttatt gctacatatc caagggtttt atatgttttt cttattatat 16860
    cttaattcaa aacaccatca cgctcttttc cagatgaaaa taaggaaaag aaattgagca 16920
    actgactgac ttaaaggtca taaaactata tagtagcaga gtcagcaaaa gaagaaacac 16980
    acatctccca agtagaggct gaaaaccagt accattcacc tccagggtga gctatataca 17040
    gattacaaag tcaccttctc taaatgttca aactgaatcc catacccata ctttaccact 17100
    acctcgtaag aacagcctca gatcttgtta tagccttttt tttagcatgc tgaagccaat 17160
    aaaatgcttc ccattcagca agagaaacaa gttctgaaac actgaataat ctgcccaggg 17220
    cctatgaaca tttccactgt gagaaatgtt ctccactgtg tggagaagat ccttactctt 17280
    ctccacacag gcagaacatt agaaaaattc ttggattcta tgatgcacag cttaggagtc 17340
    tgtttagcac aatttaagtc caaatagtta ttaaatcctc ctctgttcca gaaacagtgc 17400
    taaatactgt gaatataaaa attgaaaaga tactctcctg gctcccaaga aagtcagcca 17460
    gatagaggag acacaggcac acaaatcact gtcacatgaa gctctacctc cctaacttca 17520
    aacgagggcc taagtcacca agaatacagt agcagttgtg actacgagta actactataa 17580
    ttcaatactt tatcttccct tagaaaactc ttctcccttg gaaatttatt tgcatttcta 17640
    aataccattc cttactaaaa ggaagcaggg ctccttgggg aaatagctga ttctaggtgt 17700
    ggactatgaa atgaaaatgg tgagtctggg acatcccatg ttgcccagaa atcaaggaac 17760
    tgcccaaaga ttaacagagt catgttaaat ggacctaaga gtgaaccaga aggagctcac 17820
    tttgccccgc gtggaacaat ttcaagaaaa acatgacagt aatgaattat aaaacatgaa 17880
    ttaaaataca tattggtact aaaaagagaa caaaaggatg tggctttgga taaagctctt 17940
    cttcatggaa gaataccagc taataaatgt aaaggaaatg agagaattag aaaaattatc 18000
    attttgtaaa ccttaatata ttcacctaga catgctaaaa ccactgagta aaaggctgct 18060
    tgggaagagg atgctcacat gatctcagag tttcacacca cagataattt attagataca 18120
    ggaaggaaga tgtgatcaag cttcctgtga cccccagcca ggccccacaa cactatgtgc 18180
    ctccttgtga tgtgggagct acacagcatc gcccacacag cttctcgcca aaactgtttg 18240
    aagctaatca caagggaaga actggacagc ttctgaccat gagacgctcc accagacaac 18300
    ttgcttggcc tctccaaaga aacttgcttg gcctctccaa agaaaactca gtttcattta 18360
    aaaacaaaac taattattta aaaacaaacg aaaagcaagt tgtggacttg agctccaggg 18420
    acagagcaga catacttttc cctgttcttc ccagtaagtg gtaataaaaa ccctcaacac 18480
    tagatataaa acaaatataa gaaggttctg gaaggggaag aggaggcaga ctatccaggt 18540
    gccttgaggc ccacagaaca acccagtgat gggttcactg ggtcttcttt ttgcttcatt 18600
    atctcagact tggagctgaa gcagcaggca acttcaaaac accaaggggc acagattgaa 18660
    aagccccaag aaaagcctgc cctctctagc caaaggacca ggaaggagac agtctaatga 18720
    gatggaacac atttagacag taactgccca tttaccagca ataactgagc agggagccta 18780
    gacttccagt cttgtgagga cgtaccaagg tacccaacac ccccaccaag gctgagtaag 18840
    gactgcgact tttatccctg catggcagta gtaaggagcc catccctcac ccgccagcag 18900
    tgtcagggga acctggactt ccactcccac ccaggagtga tgaggccctc cctgctgggg 18960
    tcatgtcaga ggaggcctag tggagattca gtgacttaac cttttcccag agataatgag 19020
    gccacctttc ctccctcttc ccccatggtg acagtgaaag cactgtggca agcagtaggc 19080
    actcctaccc ctcctagcca gggaggtatc agggaggcca agtagggaac cagaataccc 19140
    acaaccaccc agcagcaaca ggggtccccc accccattgg gtgtcaatgg aagcagagcg 19200
    gaaagcctgg atatttaccc ccatctagaa gtaacaagct gatgtccccc ttcttctact 19260
    acaatggtgt tcaaaacagg tttaaataag gtctagagtc tgataacgta atacccaaat 19320
    cgttgaagtt ttcattgagg atcatttata ccaagagtca ggaagatccc aaactgaaag 19380
    agagaaaaga caattgacag acactagcac taagagagca cagatattag aactacctga 19440
    aaggatgtta aagcacatat cataagcctc aacaggctgg gcgcggtggc tcacgcctgt 19500
    aaccccagca ctttgggagg ccgaggcagg tggatcacaa gatcaggaga tcgagaccat 19560
    cctggctaac acggtgaaac cccgtctcta ctaaaaatac aaaaaaaaat agcaaggcat 19620
    ggtggtgggc acctgtagtc ccagctactc gggagcctga ggcaggagaa tggcatgaac 19680
    ctgggaagag gagcagtgag ccgagatcgc accaccgcac tccagcctgg gcaacagagc 19740
    aagacttcgt cccaaaaaaa aaaaaaaaaa aaaaaaaagc ctcaacaaac aactacaaac 19800
    gtgcttgaaa caaatgaaaa aaaaatcttg gcaaagaaat aaaagatata tattttggcc 19860
    aggtgcagtg gctcacagcc tgtaatccct gcactttggg aggctgaggc aggcggatca 19920
    cctgaggtca ggagtttgag accagcctga ccaacatgga gaaaccccgt ctctactaaa 19980
    aatacaaaat tagccagtca tggtggcaca tgcctgtaat cctagctact caggaggccg 20040
    aggcaggaga atcgcttgaa ctcaggaggt ggaggttgcg gtgagccgag atcccgccat 20100
    tgcacattgc actccagcct gggcaacaag agcaaaactc catctcaaaa aaatagatac 20160
    atattttaat ggaaatttta gaattgaaaa atacagtaac caaattgaat ggaaagacaa 20220
    catagaatgg agggggcaga caaaataatc agtgaacttc aacagaaaat aatagaaatt 20280
    acccaatatg aagaacagaa agaaaataga ctggccaaaa aataaagaag aaaaaagagg 20340
    agcagcagga ggaatgatgg aaaaagagaa aggaaggaag gaagggaagg agggagggaa 20400
    ggagtgaggg agaaagtctc aaagacctct gagactaaaa taaaagatct aacacttgtc 20460
    atcagggtcc aggaaagaga caaagatggc acagctggaa acgtattcaa aaaataatag 20520
    ctgaaaactt cccaaatttg gcaagagaca taaacctata gattcgaaat gctgaacccc 20580
    aaataaaaag cccaataaaa tccacaccaa aatacatcat agtcaaactt ctgaaaagac 20640
    gaaaagagaa aacgtcttga aagcagtgag tgaaacaaca cttcatgtat aagggaaaaa 20700
    caattcaagt aacagatttc ttacagaaat taaggaagcc agaaggaaat gacacaatgg 20760
    ttttcaagtg ctgaaagaaa agaagtgtca acacaaaatt ctagattcag taaaaatatc 20820
    cttcaagaat caatgggaaa tcaagacagt ctcagataaa gcaaaataag agaatatgtt 20880
    gccagcagat ctcccctaaa ggaatggcaa aaggaagatc atgcaacaga ccaaaaaatg 20940
    atgaaagaag gaatccagaa acatcaagaa gaaagaaata acatagtaag caaaaataca 21000
    tgtaattaca ataaaatttc tatctcctct taagacttct aaattatatt gatggttgaa 21060
    gcaaaaatta taaccctgtc tgaagtgctt ctactaaatg tatgcagaga attataaatg 21120
    gggaaagtat aggtttctat acctcattga agtggtaaaa tgacaacact gtgaaaagtt 21180
    acatacacac acacacgtaa gtatatataa atatatgtgt gtatatgtgt gtgtatatat 21240
    atatatacat ataatgtaat acagcaacca ctaacaacac tatacaaaga gataataacc 21300
    aaaaacaatt tagataaatt gaaatggaat tctaaaaaat attcaaatac tctacaggaa 21360
    gacaagacaa aaagagaaaa aaagaggagg acaaactaaa ttttttaaaa acataaataa 21420
    aatggtagac ttaagcccta acttatcaat aattacataa atgtaaatga tctaattata 21480
    tcaattaaaa gacagagata gcagagttaa tttaaaaaca tagctataag aaacctgctt 21540
    tgggctgagt gcagtgactc acacttgtaa tcccagcact tcgggaggcc aaggcgggtg 21600
    gatcacctga ggtcaggagt tccagaccag cctggacaac atggtaatac cccatctcta 21660
    ctaaaaatac aaaaaaatta gccaggcatg gtggcacacg cctgtagtcc caactactca 21720
    ggaggctgcg acacaagaac tgcttgaacc cgggcagcag aggtagcagt gggccaagat 21780
    tgcgccactc cagcctgaac gacagagtga gactccacct cagttgaaaa acaaaaaaga 21840
    aacctgcttt aaatatacca acatatgttg gttgaaatta aaagaataaa atatatcatg 21900
    aaaacattaa tcaaaagaaa ggagtggcta tattaataac ataaaataga cttcagagaa 21960
    aagaaaattt caagagacag gaataaaagg atcaagaaaa gatcctgaaa gaaaagcagg 22020
    caaatcaatc attctgcttg gagattcaac accctctctt aacaactgat agaacaacta 22080
    gacaaaaaaa tcagcatgga gttgagaaga acttaacacc actgaacaac aggatctaat 22140
    agacatttac ggaacactct acccaacaat agcaaaataa acattctttt caagtattca 22200
    ctgaacatat ccttagaccc taccctgggc cataaaacaa agctcactag tgattgccga 22260
    aggcttggat ggacagtgga agagctgcat ggggagggag aaggtgacag ttaaagagtg 22320
    taggatttct ttttgggata atgaaaatgt tccaaaattg attgtggtga tgttggcgca 22380
    actctacaaa tataaaaaag gccattgaat tgtacgtttt aagtgggtga aacatatggt 22440
    atgtggatta tatctaacgc tttttaaaaa cttaacacat ttcaaagaat agaagtcata 22500
    cagagtgtgc tctactggaa tcaaactaga aagaggtaac tggaggataa cgagaaaagc 22560
    ctccaaatac ttgaaaactg gacagcacat ttctaaaatc atccgtgggt caaagatatt 22620
    catttctgat attcattttt attgtttaat gtatttttaa aaatttctta agggaaataa 22680
    actgactaaa aatgaatatg gctgggtgcg gtggctcacg cctgtgatcc cagcactttg 22740
    ggaggccgag gctggtggat cacaagatca ggagttcgag accagcctgg ccaagatggt 22800
    gaaaccccgt ctcaactaaa aaactacaaa aagtagccaa gcgcagtggc gggagcctgt 22860
    ggtcccagct acttgggagg ctgaggtagg agaatcgctt gaacacaggc agcagaggtt 22920
    gcagtgagcc aagattgtgc cactgcacgc cagcctgggc gacagagact gcctcaaaaa 22980
    aaaaaaaaaa aaaaagaata tcaaaatttg tgggacatag ttaaagcaat gctgagaggg 23040
    aaatttataa cactaaatgt ttacattaga aaagagaaaa agtttcaaat caatagtctc 23100
    cactcccatc tcaagaacac agaagatgaa gagcaaaata aacccaaagc aagcaaaaga 23160
    aagaaaatat aaaaataaat cagtaaaatt gaaaacagaa acacaataaa gaaaatcagt 23220
    gaaacaaagt actgattctt cgaaagatta ataaaattga caaacctcta gcaaggctaa 23280
    caaacaaaaa agaaagaaga cacggattac cagttattag aatgaaagca taattagaaa 23340
    caactctaca cattataaat ttgacaatgt agatgaaatg gactaattac tgaaaaaaca 23400
    caaattacca caactcaccc aatatgaaat agataattgg gatagcctga taactactga 23460
    gaaaattgaa tttgtaattt taacactctt aaaacagaaa cattaaactt aatattttat 23520
    aaatattaga taaggtaatt atacccttcc ttaacaaata aaaacgacaa attattttgc 23580
    agctaaagag atgtatgtac tgtgaaaaat atcttcagaa aaatagaact ttgtttgaag 23640
    aataaggatt taaaaaatgt ttttaactct caagaagcaa atatctgggc ccagatggtt 23700
    tcactgaaga attctaccaa atgtttaatg aagaattacc accaactcta catagcatct 23760
    ttgagaaaac tgaagagaag ggaacatctc ccagttcatt ttatgaagtg ggtgttactc 23820
    tgatactaga actgtataag gacagctact cttgacacac tgcctatggg tagctctgct 23880
    ctgcaggaac agtcagaaaa aaaaaaaaaa gaagcactgg acaagggcag tataaaaaaa 23940
    gaaaactggg ccaggtgcag tggctcacac ctgtaatctc agcactttgg gaggctgacg 24000
    ctggtggatc acctgaggtc aggagtttga gactagcctg gccaacatgg taaaaccctg 24060
    tctctactaa aatacaaaaa ttagccaggc agggtggtgg ggaaaataaa aaggaaaaaa 24120
    aaacaaaaat aaactgcaga ccaatatcct tcatgagtat agacacaaaa ctccttaaac 24180
    tccttaacaa aatattagca agtagaagca atatataaaa ataattatac accatgatca 24240
    agtgggactt attccagaaa cgcaagtctg gttcaacatt tgaaaacaag gtaacccact 24300
    atatgaacgt actaaagagg aaaactacat aatcacatca atcaatgcag aaaaaagcat 24360
    ttgccaaaat ccaatatcca ttcatgatac tctaataaga aaaataagaa taaaggggaa 24420
    attccttgac ttgataaagc ttacaaaaga ctacaaaagc ttacagctaa cctatactta 24480
    atggtgaaaa actaaatgct ttcccctacg atcaggaaca aagcaaggat gttcactctc 24540
    attgctctta tttaacatag ccctgaagtt ctaacttgtg caaaacgata agaaagggaa 24600
    atgaaagacc tgcagattgg caaagaagaa ataaaactgt tcctgtttgc agatgacatg 24660
    attgtctcat agaaaatgta aagcaactag gggtaggggg gcagtggaga cacgctggtc 24720
    aaaggatacc aaatttcagt taggaggagt aagttcaaga tacctattgc acaacatggt 24780
    aactatactt aatatattgt attcttgaaa atactaaaag agtgggtgtt aagcgttctc 24840
    accacaaaaa tgataactat gtgaagtaat gcatacgtta attagcacaa cgtatattac 24900
    tccaaaacat catgttgtac atgataaata cacacaattt tatctgtcag tttaaaaaca 24960
    catgattttg gccaggcaca gtggctcata cctgtaatcc cagcatttta ggaggctgag 25020
    gcgagcagaa aacttgaggt cgggagtttg agaccagaat ggtcaacata gtgaaatccc 25080
    gtctccacta ataatacaaa aattagcagg atgtggtggc gtgcacctgt agacccagct 25140
    acttgggagg ctgaggcacg agaattgctt gaacaaggga ggcagaggtt gcagtgagct 25200
    gggtgccact gcattccagc ctggtgacag agtgagactc catctcaaaa aaaataaaat 25260
    aaagcatgac ttttcttaaa tgcaaagcag ccaagcgcag tggctcatgc ctgtaatccc 25320
    accactttgg gaggccgagg caggcagatc acaaggtcag gagtttgaga ccagcctgac 25380
    caacatggtg aaaccccatc tctactaaaa aatatataaa ttagccaggc atgtgtagtc 25440
    tcagctactc aggaggctga ggcaggagaa tcacttgaac ccggaggcag aggttgcagt 25500
    gttgagccac cgcactccag cctgggtgag agaacgagac tccgtctcaa aaaaaaaaag 25560
    caaaataacc taattttaaa aacactaaaa ctactaagtg aattcagtaa gtctttagga 25620
    ttcaggatat atgatgaaca tacaaaaatc aattgagctg gacaaaggag gattgtttta 25680
    ggtcagtagt ttgaggctgt aatgcacaat gattgtgcct gtgaatagct gctgtgctcc 25740
    agcctgagca gcataatgag accacatctc tatttaaaaa aaaaaaaatt gtatctctat 25800
    gtactagcaa taagcacatg ggtactaaaa ttaaaaacat aataaatact gtttttaatt 25860
    gcctgaaaaa aatgaaatac ttacatataa atctaacaaa atgtgcagga cttgtgtgct 25920
    gaaaactaca aaacgctgat aaaagaaatc aaagaagact taaatagcgt gaaatatacc 25980
    atgcttatag gttggaaaac ttaatatagt aaagatgcca attttatcca aattattaca 26040
    caggataaca ttattactac caaaatccca gaaaaatttt acatagatat agacaagatc 26100
    atacaaaaat gtatacggaa atatgcaaag gaactagagt agctaaaaca aatttgaaaa 26160
    agaaaaataa agtgggaaga atcagtctat ccagtttcaa gacttacata gctacagtaa 26220
    tcaagactgt gatattgaca gagggacagc tatagatcaa tgcaaccaaa tagagaacta 26280
    agaaagaagc acacacaaat atgcccaaat gatttctgac aaaggtgtta aaacacttca 26340
    acgggggaag atatgtctct cattaaaggg tgtagagtca ttgcacatct ataggcaaaa 26400
    agatgaacct gaacctcaca ccctacagaa aaattaactc aaaatgactc aaggactaaa 26460
    cataagatat acatctataa aacatttaga aaaaggccac gcacggtggc tcacgctcgt 26520
    aatcccagca ctttgggagg ccaaggcagg tggatcacct aaggtcagga gtttgagacc 26580
    agccggatca acatggagaa gccccatctc tactaaaaat acaaaattag ctggacgtgg 26640
    tggcacatgc ctgtaatccc agctacttgg gaggctgagg catgagaatc gcttgaaccc 26700
    ggggggcaga ggttgcggtg agccaagatc acaccattgc actccagcct gggcaacaag 26760
    agcaaaactc caactcaaaa aaaaaaaaaa aaaggaaaaa tagaaaatct ttgggatgta 26820
    aggcgaggta aagaattctt acacttgatg ccaaactaag atctataagg ccagtcgtgg 26880
    tggctcatgc ctgtaattcc agcactttgg tcaactagat gaaaggtata tgggaattca 26940
    ctgtattatt ctttcaactt ttctgtaggt ttgacatttt tttagtaaaa aattggggga 27000
    aagacctgac gcagtggctc acacctgtaa tcccagcact ttgggaggcc ggggcaggtg 27060
    gatcacacgg tcaggagttc gagaccagcc tggccaacat ggtgaaaccc cgtctctacc 27120
    aaaaatataa aaaattagcc gggtgtcatg gtgcatgcct gtaatcccag ctactgagga 27180
    ggctgaggca ggagaatcac ttgaacctgg gaggtggaag ttgcagtgag ccgagattgt 27240
    gccactgcac tccagccttg ggtgacagag cgagactccg tctcaaaaga aaaaaaaaaa 27300
    aaagaatatc aaacgcttac tttagaaact atttaaagga gccagaattt aattgtatta 27360
    gtatttagag caatttttat gctccatggc attgttaaat agagcaacca gctaacaatt 27420
    agtggagttc aacagctgtt aaatttgcta actgtttagg aagagagccc tatcaatatc 27480
    actgtcattt gaggctgaca ataagcacac ccaaagctgt acctccttga ggagcaacat 27540
    aaggggttta accctgttag ggtgttaatg gtttggatat ggtttgtttg gccccaccga 27600
    gtctcatgtt gaaatttgtt ccccagtact ggaggtgggg ccttattgga aggtgtctga 27660
    gtcatggggg tggcatatcc ctcctgaatg gtttggtgcc attcttgcag gaatgagtga 27720
    gttcttactc ttagttccca caacaactgg ttattaaaaa cagcctggca ctttccccca 27780
    tctctcgctt cctctctcac catgtgatct cactggttcc ccttcccttt atgcaatgag 27840
    tggaagcagc ctgaagccct cgccagaagc agatagtgat gccatgcttc ttgtacagcc 27900
    tacaaaacca tgagcccaat aaaccttttt tctttataaa ttatccagcc tcaggtattc 27960
    ctttatagca agacaaatga accaagacag ggggaaatca acttcattaa aataatctat 28020
    gcagtcacta aacaaataag aacaagaggc tccagaagtg ggaagccaat acccagagtt 28080
    cctacaatac agtatctgaa aagtccagtt tccaaccaaa aaatatatat atacaggccg 28140
    gacatggtag cttatgtctg taatcccagc actttgggat gctgaggcgg gcagatcacc 28200
    ctaggtcagg agttcgagac cagcctggcc aatatggcaa aaccccgtct ctactaaaaa 28260
    tacaaaaatt agccaggcat ggtggtggat gcctgtaatc ccagctactc gggaggctga 28320
    ggcagggaat cacttgaacc caggaggcag aggttgcagt gagccgagat cacgccactg 28380
    aactccagcc tgggcaacaa agtgagactc cacctcaaaa aaaaaaaaaa tatacatata 28440
    tatatgtgtg tgtgtgtgtg tgcgcgcgtg tgtgtatata cacatacaca tatatacata 28500
    tatacagaca cacatatata tatgaagcat gaaaagaaac aaggaagtat gaaccatact 28560
    ttctgtggtt atgataggat ggggtatcac gggggaagta gacaagggaa actgcaagtg 28620
    agagcaaaca gttatcagat ttaacagaaa aagactttgg agtaaccatt ataaatatgt 28680
    ccacagaatt aaagaaaagc gtgattaaaa aaggaaagga aagtatcata acaatattac 28740
    tccaaataga gaatatcaat aaaggcatag aaattataaa atataataca atggaaattc 28800
    cggagttgaa aggtagaata actaaaattt aaaattcact agagaaggtt caacactata 28860
    tttgaactgg cagaagaaaa atttagtgag acaaatatac ttcaatagac attattcaaa 28920
    tgaaaaataa aaagaaaaaa gaatgaagaa aaataaacag aatctcagca aaatgtggca 28980
    caccattaat cacattaaca tatgcatact gagagtaccg gaagcagatg agaaagagga 29040
    agaaaaaata ttcaaatgat ggccagtaac ttcctagatt tttgttttaa agcaataacc 29100
    tatacaatca agaaactcaa tgaattccaa gtaggataaa tacaaaaaga accacaaaca 29160
    gatacaccat ggtaaaaatg ctgtaagtca aaaacagaga aaatattgaa agcagctaga 29220
    ggaaaactta taagagaacc tcacttacaa aagaacatca cttataaaag aaccacaata 29280
    atagaaacag ttgacctctc atcagaaaca atgaatgata acatatttga agtgctcaaa 29340
    gaaaaaaaat aaagattcct atatacgaca aagctgtctt tcaaaaatat acatccaaaa 29400
    ggattgaaac cagggtcttg aagagttatt tgtacatcca tgttcatagc agcattattc 29460
    acaatagcca aaaggtagaa gcaacccaag ggtccatcga caaataaata aaatgtggta 29520
    tatgtataca caatggaatt tattcagtat taaaaaggaa tgaaattctg acacatgcta 29580
    caacatggct aaaccttgag aacactatgc taagtgaaat aagccagcca caaaaggaca 29640
    aataccatat tacttcactt gtatgaaata cctagggtag tcaaattcag agatagaaag 29700
    taaaacagtg gttgccaagg gctgagggag ggagtaacgt ggagttattg ttgaatgggt 29760
    acagaatttc agttttgcaa gataaaaaga gttctggaga cagatggtgg tgagggtggt 29820
    acaacaatac aaatatactt tatactactg aacagtatac ttaaaaatga ttaacatggt 29880
    gaaaccccgt ctctactaaa aatacaaaaa aattagctgg gtgtggtggc gggcacctgt 29940
    aatcccagct acttgggagg ctgaggcagc agaattgctt gaaaccagaa ggcggaggtt 30000
    gcagtgagct gagattgcgc caccgcactc tagcctgggc aataagagca aaactccgtc 30060
    tcaaaaaata aaaaataaaa aaaatttaaa aatgattaag caggaggcca ggcacggtgg 30120
    ctcacaccta taatgccagc actttgggag gccgaggcag gcgatcactt gagaccagga 30180
    gtttgagacc agcctggcca acatggcaaa accctgtctc tgctaaaaat acaaaaatta 30240
    gccaggcatg gtggcatata cttataatcc cagctactgg tgagactgag acacgagaat 30300
    tgcttgaacc caggaggcag agattgcagt gagtcgagat cgcgccactg aattccagcc 30360
    tgggcgacag agcaagattc tgtctcgaaa aaacaaaaac aaaaacaaaa agcaaaacca 30420
    aaaaataatt aagcaggaaa cgagattgct gctgaggagg agaaagatgt gcaggaccaa 30480
    ggctcatgag agcacaaaac ttttcaaaaa atgtttaatg attaaaatgg taaattttat 30540
    atgtatctta ccacaaaaaa aagggctggg gggcaggaaa tgaaggtgaa ataaagacat 30600
    cccagagaaa caaaagtaga gaatttgttg ccttagaaga aacaccacag gaagttcttc 30660
    aggctgaaaa caagtgaccc cagagggtaa tctgaattct cacagaaaat tgaagcatag 30720
    cagtaaaggt tattctgtaa ctatgacact aacaatgcat attttttcct ttcttctctg 30780
    aaatgattta aaaagcaatt gcataaaata ttatatataa agcctattgt tgaacctata 30840
    acatatatag aaatatactt gtaatatatt tgcaaataac tgcacaaaag agagttggaa 30900
    caaagctgtt actaggctaa agaaattact acagatagta aagtaatata acagggaact 30960
    taaaaataaa attttaaaaa atttaaaaat aataattaca acaataatat ggttgggttt 31020
    gtaatattaa tagacataat acaaaaatac cacaaaaagg gaagaagaca atagaactac 31080
    ataggaataa cattttggta tctaactaga attaaattat aaatatgaag tatattctgg 31140
    taagttaaga cacacatgtt aaaccctaga tactaaaaag taactcacat aaatacagta 31200
    aaaaaataaa taaaataatt aaaatgtttg tattagtttc ctcagggtac agtaacaaac 31260
    taccacaaat tgagtggctt aacacaactt aaatgtattt tctcccagtt ctggaggcta 31320
    aacacctgca atcaaggtga gtacagggcc atgctccctg tgaaggctct aggaaagaat 31380
    cctcccttgt ctcttccagc ttccagtggt tctcagtaac cctaagtgct ccttggcttg 31440
    tagctatatc attcctagca accagaaaga agaaaataat aaagattatg gcaaaaaata 31500
    atgaaatcaa aaggagaaaa atggaaaaaa ataaataaaa ccaaaagcta gttctttgaa 31560
    aagatcaacc aagttaacaa accttttaac tagactgaca aaaaggaggt aagactcaaa 31620
    ttactagaat cagaaataaa agaggggaca ttactaatga gggattagaa aagaatacta 31680
    cgaacaaatg tgtgccaaca aattagaaaa cttagatgaa atggacaggt tcctaggaca 31740
    acatcaacta ccaaaattta ctcaagaaga aagagacaat ttgaatgagc tataacaagg 31800
    gaagagactg aattgacaac caagaaacta tccacaaaga aaatcccagg cccagaagat 31860
    ttcactgtga aattctttca aacttataaa tataaattaa catcagttct tcacaaactc 31920
    ctccaaaaaa aagaacagat ctctatttac aggcgatacg atctttagaa aatcctaagg 31980
    gaactactaa gacactatga taactgataa acaagttcag caaggctgca ggatagaaaa 32040
    ccaatataca aaaatctatt atatttctat acacttgcag tgaacaaccc aaaaatgaga 32100
    ttaagaaaat aattcaattt acaataacat caaaaagaat aaaaacactc aaaaataaat 32160
    ttattcaagt aagtgcaaaa cttatactct agaagctaca aaacactgtt aaaagaaatt 32220
    aaaggtttac ataaatgaaa aactatccca tgttcatgga tcaaaagact tattactggc 32280
    aatgctctcc aaattgatct ataaattcaa caaaatcctt atcaaaatcc cagatgaggc 32340
    tgggggtggc ggttcatgcc tgtaatccca gcactttggg aggctgaggc acgcagatta 32400
    cctgaggtcg ggagctcgag atcagcctga ccaacatgga gaaaccctat ctcttctaaa 32460
    aatacaaaat tagtcaggcg tggtggcaca tgcctataat cccagctact cgggaagctg 32520
    aggcaggaga atcgcttgaa cccaggaggc agaggttgca gtgagccaag atcgtgccat 32580
    tgcactccag cctgggcaac aagagcaaaa ttccatctca aaaaaaaaaa aaaaaaaatc 32640
    ccagatgact tcactgttga aattgaaaag attattctaa aattcacatg gaattgcaag 32700
    accttgagaa tagccaaaac aaacttgaaa aacacgaaca aaatatagga tgactcactt 32760
    gccaattgca aatgttacga cacagcaaca gtaatcaaga ctgtgtggta ctggcaaaag 32820
    acacatacat acatacatat caatggaata taattgagag tacagaaaca agcctaaaca 32880
    tctatggtaa gtgcttttct atttttttct tttttttttt cttttttgta gagatagaat 32940
    ctcaccatgt tgcccaggct ggtcttcaac ttctgggctc aagcaatcct cccactgtgg 33000
    cctcccaaag tgctgggata actggcatga gccaccacat ccagcccaga tgattttcaa 33060
    aaaagtcaac aagaccattc ttttcaacaa ataggtctgg gatgatcaga tagtcacatg 33120
    aaaaaaaaaa tgaagttgga ccctccatca cactaaagtg ctgcgattat aggcatcagc 33180
    caccacatcc agcccaaatg attttcaaaa aggtcaacaa gaccattctt ttcaacaaat 33240
    aggtctggga taatcagata gtcacatgaa aaaaaaaatg aagttggacc ctccatcaca 33300
    ccatatgcaa aaattaattc aaaaatgaat tgatgactta aacgtaagag ttacgactgt 33360
    aaaactctta gaaggaaaca tacgggtaaa tcttaaagac gttaggtttg acaaagaatt 33420
    cttagacatg acaccaaaag catgaccaac taaggtaaaa tagggtaaat tgtacctacc 33480
    aaaatgaaaa acctttgtgc tggaaaggac accatcaaga aatggaaagc caaaatagcc 33540
    aaggcaatat taagcaaaaa gaacaaagct ggaggcatca tactacctga cttcaaagca 33600
    acagtaacca aaacagcatg gtactagtag aaaaacagac acatagacca atggaacaga 33660
    ataaagaacc caaaaataaa tccacatatt tatagtcaac tgatttttga caatgacacc 33720
    ccttcaataa atgatactag gaaaactgga tatcgatatg cagaagaata aaactagacc 33780
    cctatctctc accatataga aaaatcaact cagactgaat taaagacttg aatgtaagac 33840
    ccaaaactat aaaactactg gtagaaaaca taaggaaaaa cgcttcagga cattggtcca 33900
    ggcaaagatc ttatggctaa aacctcaaaa acacaggcaa caaaaacaaa aatggaaaaa 33960
    tagcacttta ttaaactaaa aagctcctgc acagcaaagg aaacaacaga atgaaaagac 34020
    aacctgtaga atgggagaaa atatttgcaa actatccatc catcaaggga ctagtatcca 34080
    gaacacacaa gtgactaaaa caactcaaca gcaaaaaagc aaataatctg gtttttatat 34140
    gggcaaaaga tctgaataaa cattctcaaa ggaagacata caaatgtcac tatcattctg 34200
    ccagtaccac actgtcttga ttacttgtta gtgtataaat ttttaaattg ggaagtgtga 34260
    gtcatcctac actttgttct tgtttttcaa gtttgttttg gctattctgg gagccttgca 34320
    agtataaaat agccaacaag tatgaaaaaa tgctcaccat cactaatcat cagagaaata 34380
    aaaatcaaga ccactatgag atatcctctc actccagtta gaatggctac tatcaaaaag 34440
    acaaaatata atggatgctg gcaaagattt ggagaaaggg gaactcctat acactgtggg 34500
    tagggatgca aattggtaat ggccattatg gaaaataata ctgaggtttt tcaaaaaact 34560
    gaaaatagaa ctaccatatg atccagcaac cctactactg ggtatttatc caaaggaaag 34620
    aagtcagtat actgaagaaa tatatgcact ctcatgttaa ttgcaacact gttcacaaca 34680
    gccaagacag ggaataaatc taaatgtgca tcaacagatg aatggataaa gaaaatgtgg 34740
    catatacact caatagaata ctattcagcc attaaagaag aatgaaatcc tgtcatccca 34800
    gcaacatgga tgaacctgga ggacattata tttaatgaaa taagtaaagc acaaaaagat 34860
    aaacagtaca tgttctcact cagacatggg tgctaaaaag aaaatggggt cacagaatta 34920
    gaaggggagg cttgggaaaa gttaatggat aaaaatttac agctatgtaa gaagaataag 34980
    ttttagtgtt ctatagaact gtagggcgag tatagttacc aataacttat tgtacatgtt 35040
    caaaaagcta gaagagattt tggatgttcc cagcacaaag gaatgataaa tgtttgtgat 35100
    gatggatatc ctaattaccc tgattcaatc attacacatt gcatacatgt atcaaattat 35160
    cactctgtac ctcataaata tgtataatta ttacgtcaac aaaaaaagga aaaaaaagaa 35220
    aattaagaca acccacataa tggaagaaat aaaatatctg caaattatat atatctgata 35280
    aatatttaat atttataata tataaagaac tcctacaact caagaacaac aacaaaacaa 35340
    cccaattcaa aaatgggtaa aagccttgaa tatacactta tctaaagact atatacaatt 35400
    ggccaataaa gacacgaaaa gatgctcaac atcactagtc atcagggaaa tataaatcaa 35460
    aaccacaatg tagaatgtag acaccacttc atatgcacta ggatggctag aataaaaagg 35520
    taataacaaa tgttggtaag gatgtgaaaa aatcagaaac ctcattcgct gctgttggga 35580
    atgtaaagtg atgcagccac tttggaaaac agtctggcag ctcctcaaat tattaaatac 35640
    agagttaccg tatgacccag gaatattcct cctgggtcta taaccaaaaa aatgaaaaca 35700
    tatatccaca taaaaacttg tacatgggca tttatagcaa cattattcat aacagcaaag 35760
    gtggtaagaa cccatatgcc catcatctga tgaacaggta aataacatgc ggtattatcc 35820
    atacactaga atattatctg cccatacaag gagtgacatc cagctacatg ctacaaggat 35880
    gaatctcgga aaccttatgc taagtgaaag aagccagtca caaatgacca cagattatga 35940
    ttccatgcat cggaaatgac cagaataggg aaatctatag agacagaaag tagattagtg 36000
    gttgggtggg gctgggagga caggtagtac actactttcc cagaactact ggaacaaagt 36060
    accacaaact ggggagctta aacatagaaa ttgatttcct cacagttctg gagactagga 36120
    ctctgagatc aaggtgtcag cagagctggt tctttctgag ggccctgagg caaggctctg 36180
    tcccaggcct ctctccttgg ctggcaggtg gccatcttct ccctgcgtct tcacatcatc 36240
    ttttctctgt gtgtgcccat gtccaaattt tgattggctc attctgggtc atggccaatt 36300
    gctatgcaca aagtgaagtc tacttccaaa agaagggaag agggaacact gactaggcta 36360
    aacttatagt cattttaatg tccgcttttc ctatgagatt gtgaacacac agaagtaggg 36420
    tttttatcta cattgtgcaa agtttaataa gaaaaataga attcaagaga agcagttcaa 36480
    tagcaggaat ttaatatggg aactaattac aaggtttagg gcaggactaa aaagccagtt 36540
    gggatggtga gccaacccag agattagcaa cagtgggacc ccatctacct accacccatg 36600
    aagctggaag gataaaggag gggctattat cagagtccac aagccagtgt cagagtcctt 36660
    ggctggagct gggaccaccc tagagacact gtgcaaagca gaaaacaagg gggaaaaacc 36720
    ctgacttctc ccttcctccc acctttcaat ctcccactag tgcttcctac tagccatact 36780
    tggccagaga cagtgacaag gaacactgca aaatgaagtt tgtaggaatc atctccctct 36840
    gagacagaga aatatggaag ggtagaaaat gaatcagagg ataaagagaa aaaaccctga 36900
    gtactatctt atttatcttt gtatctccag tgcctaatct gtctctcaaa aaaggaaagc 36960
    aattgagaga aactgaaaac tccaattgaa atgaaagaat ggagaattac tggactagaa 37020
    gagaagagaa aaatttattc cgcatagagt aaacaagaat ggattcacaa aggacgtgat 37080
    gaatgaaaag ctataatcag caaagatttg ccagagaaat taaaaagtgg taaactcagc 37140
    cacgctgtac aacctgaagg cacaatgcat gaaaacgttt caagaaatga caagatttga 37200
    agtcaaattc taagtgcttt tccagaatct ctcaagacga ttatatagct accccatttt 37260
    attaaataaa atggaaactt actaaacttt ccccttgtat taaactaaca tatgtcctaa 37320
    tagcaaacga ttctggaatt cctagagtaa aatatatttc gtcaaagtgt attgctcttt 37380
    taatattctg ctgacctcct tttgctattt aggatatttg tatacacatc acacgtaaat 37440
    ttggtctata gtttacatct acgggcttat actgttcttt ttttcatttt tttaaaattt 37500
    ccaaccccca gtatccatat actgctctct atcagggtta ttttaacttt gtaaaatcag 37560
    ctgagatgct ttccatgttt ttttttttta ttttctgcca catttgaata gcataggagt 37620
    taccaccatc aaccttggat tatttaagca ttcacgattc cacgtgtgga ttttttattc 37680
    agagtctttc ttgtcattcc tgctatcagc acagaaccca atctcagctt tccagctata 37740
    ctctcacccc atggaatttg cagatgaagt tcaaaaggac ctttgcatta tcctgcctcg 37800
    ccctcttccc ccttcattta gacatcacct tcttctagaa cgtcttacct gacatgccct 37860
    gctcccaacc cctgctgccc aattgtgtgc tctcccgtgt cctggcctgc catcctcttt 37920
    agtaattgcc tgctccctca tctgtctccc cacccagaca ttaagctgaa tagactggat 37980
    ttgtgtcttg tccatcacta taatctcagc acctagtacc tagtaggtac ttaccatgta 38040
    ttcattagca aaatgttatg tataaccttg caccttaaaa acaagagaag gaagacaaaa 38100
    ttaagtctta agactatggt ttagaacatg gatcagaaac tacagtctgc agcccaaatc 38160
    cagaccaaat gaagagacca tgttcattta catacaacct atagcagctt tcacactaca 38220
    ggagcagagc taagtagttc caagggaaca cacggccctg caaagcctaa aatatttact 38280
    ctatagctct tcacagaaaa agttttcaga tccctcgttt agaactcttg ttcatatgca 38340
    atttcactaa accatagttt tttgggtttg tttggttttt tttggcaaaa aggaatgagc 38400
    cgatccagaa aaggttgaaa agaatgaatc attactgctg aaagaatgtg cacacagtcc 38460
    gtcagtattc tgctgccatg ctgacaccca tccaatagtg tcatgagatg cagcagctac 38520
    tactgtgttc tcaatgccga gtccacccac tccataacca tgtccaagca atcttgggaa 38580
    catcatcacc atgcttgttt atccttaagg tattgcctca catacagcag tggctggtca 38640
    taaagtcaaa tgacactagt ggccaggagg tcaagagaat gagtgaggac aggtgggtag 38700
    gcagcccagg ccctagcaac agcaggagct cacccctcag tcactctagc caggactgaa 38760
    atacttttca ccctttcaag agagactagg aatctggatt tttatgtgaa atatcttgat 38820
    tactaaatgt tgtcaacaga catgtcaaaa ggtaaaacta agtaagttca tggggcagat 38880
    tgactattca ggttatagaa ttaaggattc ttatccaaca cagataccaa ccaaaaagct 38940
    gacgtataac atattaggag aaactatgtg cactgtcgaa acatcaacaa ggggctaatg 39000
    tctaaaatag tctatattgg attccagttg aaacatgggg aaaggacatg aacaggcaac 39060
    ttatgtcaat ggaaactcaa aaagataaca agcatatata aaagcattct caaattcagt 39120
    agtaaacaga cagatgcaaa taaaaagagg gaaactgctg ccgggcacag tggctcacac 39180
    ctgtaatccc agcactttgg gaggccgagg cgggcggatc atgaagtcag gagatcgaga 39240
    ccatcctggc taacatggtg aaaccccgtc tctactgaaa acacaaaaaa ttagccaggc 39300
    gtagtggtgg gcaccagtag tcccagctac tcaggaggtt gaggcaggag aatggcatga 39360
    acccaggagg cggagattgc agtgagccga gaccatgcca ctgcactcca gcctgggcga 39420
    ctgagtgaaa ctccatctca aaaaatataa taataattat aattataata ataataaata 39480
    gtaaataaat aaaaagagag agactgctaa agtctagaaa gttgaatgat gccaagcgca 39540
    tgcaaagatc agggccttgg gatggccggg tgcagtggct cacgcctgta atcccaccac 39600
    tttgggaggc caaggcgggc ggatcatgag gtcaagagat caagaccatc ctggccgaca 39660
    cagtgaaacc cggtctctac taaaagtaca aaaaaatata tatatatata tatattatta 39720
    tattatatat atatatatca gagccttggg aatccttgtg tgctgctggg gaaggtagtg 39780
    gtgcagccac ccttgacagc aatctggcag tacttggtta tattaagtat aggcacacac 39840
    cacgaccagg cagtcctact cctgggtcta aatcccaaag aattctcaca caagtccata 39900
    aggagacatg tacgaggctc attcagcatt actgggagtg ggaatcaacc tgggtgtcca 39960
    tctacaggag acgagatgga caaaatgtgg tggatattaa gaccagaatc accaagtaac 40020
    agagatgggt ggtgagtgac aatcctaaga tacagaataa aggctagaac atgatgccat 40080
    tcatgtaaat taaaaataga tgcacacaaa gcagtatacg cgtgaccctt gaatagcaca 40140
    ggtttgaact gcctgtgtcc acttacatgt ggattttctt ccacttctgc tacccccaag 40200
    acagcaagac caacccctct tcttcctcct ccccctcagc ctactcaaca tgaagatgac 40260
    aaggatgaag acttttatga taatccaatt ccaaggaact aatgaaaagt atattttctc 40320
    ttccttatga ttttctttat ctctagctta cattattcta agaatatggt acataataca 40380
    catcacacgc aaaataaatg ttaattgact gtttatatta tgggtaaggc ttccactcaa 40440
    cagtaggctg tcagtagtta agttttggga gtcaaaagtt atacacagat tttcaactgt 40500
    gcaggcaatc agttcccctg accccctcat tgttcacggg tcaactgtat atacacaaaa 40560
    gtattatatg aacctcatta gaatagctgt ctatagggag aagagaatga gagtgggata 40620
    aaacggaatg aacaaataaa ccaacaaatg cattaacaag caaaacaaca gaggggcttg 40680
    catgggccag tgatgataaa gggctaagaa tgagaatata attaattcaa ttcctcacac 40740
    ctgaggtcta aaaccaagga aagggagggc caggcgtgga ggctcacgcc tgtaatccca 40800
    gcactttggg aggctgaggc gggcggatca caagattagg agtttgagat cagcctggcc 40860
    aacacagtga aagcccatct ctacaaaaaa tacaagaatt acccaggtgt ggtggcacat 40920
    gcctgtagtt agctactctg gaggctgagg caggagaatc acttgaaccc aggaggcgga 40980
    ggttgcaggg agccgagatc acaccattgc actccagcct gggtgacaga gtaagactct 41040
    gtctcaaaaa aataaaaaaa ataaaaaaac agagaaaggg aggaaactag atccaggctg 41100
    actagataca gcctttagag ttagaaaaga tgatttgaca atctaagccc acactcagat 41160
    tgaatgaaat tgaaaagcct ttcaaactaa aacatttaat tacaccatct gctgcagaca 41220
    gaactcagac aactcaaaca ggtaatgtca gcgtggtgtt ttatatcacc accctcaaca 41280
    cagaataaaa atcagctgca tgtgaagcag tgactagaat gaagaaaagg ctgcttctta 41340
    cttccttcta gtggttcttt ccgaaaacat taataggcac cagctctatg catgtcaccc 41400
    tgcagggaga catggggtat ataactatga cttactgttc attcctcaag gaattcccaa 41460
    tcttgtggaa gattatacac aatgaggcaa caaaaactat ccaataaaac cacggaaaag 41520
    aagccagtga caaagaagcc agtgatgaaa ggccctgtga gcagagctga tggccatttg 41580
    gggaagaaag accaacatgg atgggggtga tcagggtggc tccgtgggaa agctggaaga 41640
    gaagtggcag atctctgagc tggatgatgg gccactacca tctgtatatg gctaattaaa 41700
    gaccatgtgt ggatttttta ttcagctctt tcgtgtcatt cctgctatca gcacagaacc 41760
    caatctcaac tttccagcta tattgagcta aacttctcac ctcatggaat ttgcagataa 41820
    agttcaaaag gatccttgcc ttttcaaaat aattttgaat ggttgagtag tccctctgtg 41880
    ctctctcact gacaccctct caaggctgct gagcacgtgc catgctatgg ctttctccaa 41940
    catcaggaaa tgttctccac tcagtttcac cttaatacaa atgtgttctc tcttcagaga 42000
    aggcaaaaaa attcatgacc atctgactgg gagaagtcat ttctaggtaa agtgtccatc 42060
    tttttctgag gaacacagga ggaaaatctt acagaaaaga gttaacacag caggcctaag 42120
    actgcttttt aaaataaata aataaataaa taaataaata aataaataaa taaataaata 42180
    aataaatgaa tgatagggtc ttctgtattg gccaggctag tctcaaattc ctggcttcaa 42240
    gagatcctcc caccttggtc tcccacagtg ttgggattat agacatgagc cattgtgctt 42300
    ggcccaagac tgttattctt aaaaagtctc ataaaaagca tggttaatcc ttggctggca 42360
    cctgggaact tagatttcag aagggttccc accatccaac ctggaaagag ggactcactg 42420
    tgcctaaatt attgtgtggt ttatgctgaa ctcctgcttt tcttcaggta gcgtggaatg 42480
    tggtatgtgc tgggcaaagg gggcctgcat gaccagcccc caataaaaac cctgggtgtt 42540
    gggtctctag tgagtttccc tggtagacag catttcacat gcgttgtcac agctccttcc 42600
    tcggggagtt aagcacatac atcctgtgtg actgcactgg gagaggatgc ttggaagctt 42660
    gtgcctggct tcctttggac ttggccccat gcacctttcc ctttgctgat tgtgctttgt 42720
    atcctttcac tgtaataaat tacagccgtg agtacaccac atgctgagtc ttccaagtga 42780
    accaccagat ctgagcatgg tcctgggggc ccccaacaca gaaataaatt ataaaagacc 42840
    aaggactggg catggtggcc catgccggta atctcagcgc tttgggaggc cgaggcagga 42900
    ggaccagtta agcccaaaag ttcaaagtta cagtgaccta tgactgcgcc aatgcactct 42960
    aacctgggag acagagcaag accctgtccc caaaacaata aactaaacac atacttctgc 43020
    cttccaagtg tcttaaaatt caatggaatg gtagaaacat ttttaaaaca ctaaatcaaa 43080
    agaaacctgg aaaacaagag tgccgatggc caactaaaat gtctaggaaa tttctgaaaa 43140
    gtaaaaagta ctcagaacca gattacctga gcaaaccata gcccaataca agcttgggag 43200
    gaggctgtta tgcagaagga aatggtaaca ggtttccagg aacagacttg taacagcaga 43260
    tagaacagca gaggtagaac ctgacaaggt gattacctgg ggaactgcag tctgaatgac 43320
    caggactgtt ggacccttcc cctcacatgg aatacacacg ccactcagca gcacaccaca 43380
    gctcttcaac aatcacagga ggcacgctac gcctagtaag acaggaaaaa aggaattctc 43440
    aaacttcgaa gatgaacaca taaagaatca ccaagttttt attcagtatg atgaaacagg 43500
    gacactgaat caacagaaca caaacccaag caaagataat tactagagca catagaagaa 43560
    attattagat attcttggga agacctaagg ggacattata aagagcaagc agttggtatg 43620
    tgacgatctt tgtgatatac caagaaataa aaacacagga tgaagaccag atagagaata 43680
    atgctactat ttgtgcaaaa aaggagaaat ggagaatctg attcatattt gcttgtattt 43740
    gcatgaagaa actttggaag gtacataagt aactaacaac aatggttacc tacttgtaag 43800
    gcgagagaag taagaggaca ggaatggtgg gaacaccttt tgtgtccgga attggtgggt 43860
    tcttggtctg acttggagaa tgaagccgtg gaccctcgcg gtgagcgtaa cagttcttaa 43920
    aggcggtgtg tctggagttt gttccttctg atgtttggat gtgttcggag tttcttcctt 43980
    ctggtgggtt cgtagtctcg ctgactcagg agtgaagctg cagaccttcg cggcgagtgt 44040
    tacagctctt aagggggcgc atctagagtt gttcgttcct cctggtgagt tcgtggtctc 44100
    gctagcttca ggagtgaagc tgcagacctt cgaggtgtgt gttgcagctc atatagacag 44160
    tgcagaccca aagagtgagc agtaataaga acgcattcca aacatcaaaa ggacaaacct 44220
    tcagcagcgc ggaatgcgac cgcagcacgt taccactctt ggctcgggca gcctgctttt 44280
    attctcttat ctggccacac ccatatcctg ctgattggtc cattttacag agagccgact 44340
    gctccatttt acagagaacc gattggtcca tttttcagag agctgattgg tccattttga 44400
    cagagtgctg attggtgcgt ttacaatccc tgagctagac acagggtgct gactggtgta 44460
    tttacaatcc cttagctaga cataaaggtt ctcaagtccc caccagactc aggagcccag 44520
    ctggcttcac ccagtggatc cggcatcagt gccacaggtg gagctgcctg ccagtcccgc 44580
    gccctgcgcc cgcactcctc agccctctgg tggtcgatgg gactgggcgc cgtggagcag 44640
    ggggtggtgc tgtcagggag gctcgggccg cacaggagcc caggaggtgg gggtggctca 44700
    ggcatggcgg gccgcaggtc atgagcgctg ccccgcaggg aggcagctaa ggcccagcga 44760
    gaaatcgggc acagcagctg ctggcccagg tgctaagccc ctcactgcct ggggccgttg 44820
    gggccggctg gccggccgct cccagtgcgg ggcccgccaa gcccacgccc accgggaact 44880
    cacgctggcc cgcaagcacc gcgtacagcc ccggttcccg cccgcgcctc tccctccaca 44940
    cctccctgca aagctgaggg agctggctcc agccttggcc agcccagaaa ggggctccca 45000
    cagtgcagcg gtgggctgaa gggctcctca agcgcggcca gagtgggcac taaggctgag 45060
    gaggcaccga gagcgagcga ggactgccag cacgctgtca cctctcactt tcatttatgc 45120
    ctttttaata cagtctggtt ttgaacactg attatcttac ctattttttt tttttttttt 45180
    tgagatggag tcgctctctg tcgcccagac tggagtgcag tggtgccatc ctggctcact 45240
    gcaagctccg cctcccgggt tcacaccatt ctcctgcctc aacctcctga gtagctggga 45300
    ctacaggcaa tcgccaccac gcccagctaa ttttttattt tatttttttt ttagtagaag 45360
    cggagtttca ccatgttagc cagatggtct caatctcctg acctcgtgat ccatccgcct 45420
    cggcctccca aagtgctggg attacagacg tgagccactg cgccctgcct atcttaccta 45480
    tttcaaaagt taaactttaa gaagtagaaa cccgtggcca ggcgtggtgg ctcacgcctg 45540
    taaccccagc actttgggag gccgaggcgg gcggatcacg aggtcaggag atcgagatca 45600
    tcctggttaa cacagtgaaa ccccgtcgct actaaaaata caaaaaatta gccgggcgtg 45660
    gtggtgggca ccggcagtcc tcgctactgg ggaggctgag gcaggagaat ggcgtgaacc 45720
    tgggaggcag agcttgcagt gagccgagat agtgccattg ccttccagcc tgggcgacag 45780
    agcgagactc cacctcaaaa aaaaaaaaaa aaaatagaga cccggaaagt taaaaatatg 45840
    ataatcaata tttaaaaaca ctcaagagat gggctaaaga gttgacggaa caaatctaaa 45900
    tattagattg gtgacctgca aaaccagccc aaggaacatc ccagaatgca gcccataaag 45960
    ataaagagag catttccgct gggcacagtg gtatggcagg ggaattgcct gagtccaaga 46020
    gttgcaggtc acattgaacc acaccattgc actccaggcc tgggcaacac agcaatactc 46080
    tgtctcaaaa aaaaaaaaaa ttaaattaaa aaagacagaa tatttgagag aaaaaaatgc 46140
    ttatttcaag aaacatgaaa gataaatcaa gatattctaa ttcccaagta agaataattc 46200
    cagaagcaga aaatagaata gaggcaagga aacactcaaa acttctccag tgccatagaa 46260
    atgtgtatta atctttagaa tgaaacggac taccaaatgc tgagcaggaa gaacaaaaga 46320
    gatccactct taagccagtg tggtgcccaa gcgcagtggc tcatgcctgt aatcccagca 46380
    ctttgggagg ccgaggcagg tggatcacct gaggtcagga gtttgagatc agtcaggcca 46440
    acatggtgaa accctgtctg tactaaaaat acaaacatta gctgggtatg gtggtgcaca 46500
    tctgtaatcc caactacttg ggaggctaag gcaggagaat cacttgaaac caggaggtgg 46560
    aggttgtagt gagccgagat catgccacac tcccagcctg ggtgacagag caagattcca 46620
    tctcaaaaaa aaaatccact cctagacaaa taatagttaa attttagaac accaaggaga 46680
    aagaaaaaaa attgtaaagc ttcagagaaa ataaacatta actacaaaga aacgagagtc 46740
    agacgcgtgc acttcttcct agataccagc agataaagca atatctccaa aattcagaag 46800
    gttttaacgt agaatcctat acccagtcaa gaatattcac atggaaaagt gaaataaaaa 46860
    acattgttta aacatgcaag ggttcagaaa gtttaccatt cacagaatcc ctgaaaacaa 46920
    aaccaaataa tcacttaagg actcattaag aaaacaaatg aaataaaagc accaatgatg 46980
    agtaaataat cagaaaaatt tacagtttac ctaaataact gtttatgcat aatgtatgaa 47040
    aacccaaaaa tttaatatgg gacagaatta aaatcatgat aagattcttt tttgctttac 47100
    tcatggagag ttcacataaa cagattatct tttaatagca agagaaaaaa atgtttagat 47160
    atgtgtgaaa aactaagggt accaaaacag tgcaaattca tttatcatca ggaaaatcca 47220
    aattaaaacc acagtatcca ccagaataac taaaaggtaa aagacagaaa ttaccaagag 47280
    ttggcaagaa tgtggagcaa ccacatatac ttctggggta aataagttgg tgcaaccggt 47340
    actgaaaact gtttgctagt atctactaaa accgagcaca tgcacagact acaaccaagc 47400
    agttccactc ccagatacac actcaacaga aatgcacaca ctcactcaac aaaagacgtg 47460
    tactagagtg ttcatgtact tactattcat aatagtccaa aaatgcaaac aaccaactgc 47520
    caatcaaagt caaatgtata tctatattag ggatatatac aatggcatat acacagcaat 47580
    gagaatgaaa tgaaccagct cggcacagtg gttcatgcct gtaatctcag cactttgggc 47640
    gggtaaggca ggcagatcac ttgaggtcag aaatttgaga ctagcctggc caacacggtt 47700
    aaaacctgtc cccactaaaa acacaaaaat tagccgggca tagtggttgc aggcctgtaa 47760
    ttccagctac tcgggaggct gggttgggag aatcgtttga acccgaaagc cggaggtcgc 47820
    agtgagcgga gatcgtgcca ctgcactcca gcctggacga tagagcaaga ctccgtctca 47880
    aaaaaggaaa tcaaaaatat aaaataagat gacaggaata atccgcaaaa gatcagtaat 47940
    caaaataaat ataaatgggc taaagctacc tattaaaaga caaagatttc acacccataa 48000
    ggatagctac tatcaaaaaa agagagagaa taacagatgt tagcaaggat gtatggaaac 48060
    tgaaattctc acgcattgct ggtgagaata taaaatggtt cagcctctgc ggaaaacact 48120
    atgctgggtc atcaaaaaat taaaaataga agtactactt gatccaacaa ttctacttct 48180
    gggtatatac ccaaataact gaaagcaggg tcttgaagag atatttgtac acccatgatc 48240
    atggcagcat tattcataat agctatgatg tggaaccaac ataaatatcc tttgataaat 48300
    atatggataa gcaaaatgtg gtgtatacat tcaatggaat attaattagc aataaaaatg 48360
    aagaaaattc tgacacatgc tacaacatgg atgaaccttg agggcattac attaaatgaa 48420
    ataagccagt tataaaaaga caaatactat atgaggtact atattagata ctcatgcaag 48480
    gtacctaaaa taggcaaatt catagagaca aaaagcagaa tggtggttgc caggggctgc 48540
    ggtaatggat acagagcttc aattttgtaa gatgaaaaaa ttctggagat tggttgcata 48600
    acaatgtgca cacacttaac actggggaac tgtaaactta aaagtagtaa atggtaaaaa 48660
    taaaaataat aaataataaa ttttatgtta ttttaccaca atatttatta aaagacaaag 48720
    attaactaat taaacaaaat ccagccataa gctaatggta agagtaacaa ttaaagaaga 48780
    cacagaaaat tgaaaatcag tgactagaaa aagatattcc atataaatgc taacaaaaag 48840
    caagtacagc aatataaaga gaatgaacaa aaaaaaaatt aaataagatg gctcgtttat 48900
    tcccaaaagg tacaattcac caagaagata caagaattgt gaacctttaa gcacataaaa 48960
    cagcttcaaa aatacaacat ttaaagaaaa atatatatta aacatagaaa tagtacaaaa 49020
    acccctacaa gaatcataat gggagtcttc aatacaactc tccatatcaa caggtcaaac 49080
    agagaaaaaa aataagttaa ggatgcagaa aacctgaatt accatcaata aacttgagat 49140
    taatatagaa ctgtataccc aatatactaa gagttcaggg aacagtcgtg actgacagtg 49200
    gactgcaaat taatctgttc ttaatctttg tttttctttc agcactgtgg cagaatagag 49260
    atcctaaaaa ccttccagct acaaaacatc tttttaaaaa tataaaaaaa tacaaaaata 49320
    actctgaaat caatagaaga cacatggtga aaccaaaatt ctagaataca gggagaataa 49380
    aggcattttc agatattaca aaaacagaaa attgatcatt gctgaagtaa tttctaaaga 49440
    atgtacttga gggagaagaa aaatgttcca aagaaaagta tctgtgatac aagaaggaat 49500
    ggaaagtgaa gaaatggtaa acaggtagat aaagctaata aatgttgacc tagaaaataa 49560
    caaaaacaat agcaataatg tctcgttgga agggttgaag taaaaataca attaaggcca 49620
    aatgtgaggt aagtggaatg aaagaattag aagtccttgc cttgttcaca ggactgatta 49680
    aataaatgag ccaggttttc cattcaaaca gttaaaactt gaacaaaata aactcaaatt 49740
    aagtagaaag ataaaaaaca gaaattaatg tcatagaaaa ataaaaaatc aatagaatta 49800
    atcaataaat cctggttaat aaaagctggt tctttgaaag gattaataaa ataatcatta 49860
    agcaagtctg atcaaaaaaa aagagaaaag gtaccaaaaa aagtactgta tcagaaagag 49920
    aacatacaga tacatacaga tatgtaagag tctgttttct tacaccagaa tactatatac 49980
    aacattatgc tagcatatat taaatttcaa taatgttaat gattttctag gaaaacagaa 50040
    aatattaaat ttactttgaa gaaacagaaa aactgagaaa aataaatgat catgaaaaaa 50100
    atgaaaaggt aattaaatac tgatattaac tgcctaaaca acaccagcag cagcccaggc 50160
    agtctgcagt caagttctgc caaacttgag ggaacagata attcttctat tccagagcat 50220
    agaaaatgat ggaaagtttc ccaatttaat cagagaggac agcctgatcc ttgttatgaa 50280
    cacagataaa aatggggtaa actatatgcc aaactcagat accaaaaccc taaataagat 50340
    gctagcttat tgatgtgaac aatccaaaag tgcattttaa attagcccag ggttttagag 50400
    aaagaaaatc tagcaatgtg accaccactt atgttaacaa ttttaagacg aaaatctaca 50460
    tgatcatatc aatgcatgct acacaaaagc atttgggcaa aaaacccaac acccaccctt 50520
    gactttttaa actcttagta attaggcata aacagaaatg tacttaatgt gatagaatac 50580
    actcggtgaa gatacagagg gaatgctccc taaaaccaag cccaagacaa agattcctat 50640
    ttaacctcaa tagtcaacac tgcagcgaga gtaatctatg gaagacaagg aaaaaagtaa 50700
    aaacatgaga gacatctgtt gtttaacaga caataagatc acctacttgg aagaggcaaa 50760
    cgaatcaagc gaaaaactat taaaactgag acaggcttta gtatggaggc tcagcttcag 50820
    ctgtagtttg ggctaccaaa ttcaactcgc ttgcttggag agttaatcct gcaaagctaa 50880
    tttctgttga ggtattagga ttgacaagcc tgtgctcctc cctcctcccc catcttcaac 50940
    actgaaataa cacggtgttt ggaactggat aacagaatct tccaaaaaca aaaattgtcc 51000
    tgaagggctg acttgtgccc ttactcaaaa aacactttat ctgctgcctg cagctcctac 51060
    agttgctggt ggataagcct gccaaccagc tcggcgtaat tcttcctgca gagggcaagg 51120
    aagagcactt tcacaggaaa atttttttcc gaactgtatg ccgcttatta cataaactta 51180
    cgtgctggca aatggagctc cagcaaaata agatattcag agtcaaactt ccttaggaaa 51240
    aaaaaaaaaa aaaagcaagc acataacact aatttccttg catgggcact ggggaaggag 51300
    gtcgttactt ccgcacgccc gcaggtccgc accaccggga aacccacggg caccgcgcgc 51360
    tgcccccggg ccttccaggt gcactgcgcc gcggcgcccc agctgacccg ggatgcgcag 51420
    ccctagccct tcccctgtca ccccggccag gaaggggcgg gagcgcggcg gacgccgagg 51480
    gcgaagggct tctcggtcct ctgcaccacg cagcaccccc aaggcacaac agggagggtg 51540
    cgggaggctc ccgagaccca ggagccgggg ccgggcgtgc ccgcgcacct gtcccactgc 51600
    ggcgagggct ggggtcgcct ccagggccgc agctgtcggg agccacctgg ctctcagtcc 51660
    cgggtccctg cgacaaccct cgggcccgga ggggaggagg cggccacctg ccgctgccac 51720
    ctgcggcacc ggtcccaccg ctccgggccg ggcaggacag gccaggacgt ccctcctggg 51780
    ctggggacag gacacgcgac gaggggaccg gggcccccgc ggcgaagacg cagcacgcct 51840
    tcccagaaag gcagtcccgt gcccccacga cggactgccg gacccccgcg ctcgcccgcc 51900
    catcccttca gaccacgcgg ctgaggcgca aagagccggc cggcgggcgg gctggcggcg 51960
    cggctagtac tcaccggccc cgctggctca gcgccgccgc aacccccagc ggccacggct 52020
    ccgggcgctc actgatgctc aggagaggga cccgcgctcc gccggcgcct ccagccatcg 52080
    ccgccagggg gcgagcgcga gccgcgcggg gctcgctggg agatgtagta cccggaccgc 52140
    cgcctgcgcc gtcctccttc agccggcggc cgggggcccc ctctctccca gctctcagtg 52200
    tctcatctcc ctatctgctc atcctctggt cgcacataat cgatgtttgg gcgtcccaag 52260
    ccagatgtgg accccatttc cgcactctac actggaggtt ttctaagggt ggtgcccgga 52320
    ccagcagctt cagcctcatc tgggaacttg agaaaatgca gattctccgt cccacccagc 52380
    ctattcggtt tttcctgcac taaaaccatg aaggtggggc ccagcagtcc acattctcgc 52440
    aagcccgtca agtgattctg aggcgccctc cagtttgaga gctatgctca cggcctcacc 52500
    tccgccccgc aaggagcccg gtcttgcctg tggcgctagc cgcacacgga cacctcatcc 52560
    tgcggggccc gcccccccgc tgcaccctca ccgcccaacg cctcctccgg gatgcagcgg 52620
    aggcgcctgg aagtcggcaa ggtcaacatc cccctcagca tcttccctac cctcacggct 52680
    cctcctccag gggtgcctca tggccagggg ttagaaagag ccactgtgtt tcttgacatg 52740
    gaagtggcct aagaccttaa tgaaaactgc aggagtggaa tgacagaacc tttggtcata 52800
    cttgagggcg tgaagctcaa atgaggagga aggaaaggat ccagggagaa taaccaaccc 52860
    tggcaagttg tggcgcccag gtagaggggc gagcctaggc tagcggttct cgaccagggc 52920
    cggtgttgcc cctcctcgcc gccccgcgta catttgggga ggtctggaga catttttggt 52980
    tgtcatgatg cgggagttgc tactgttgcc taagtgggta gacacgaggg tgctcctcaa 53040
    catcctacct gaaggacagg actgccccac aaggaagaat gatccggccc caaataagaa 53100
    accctgggct ggtcagcaac aacccctttg ttctgagaag agaggaggaa agaataaaag 53160
    aagtggggtg aagttttggt ttggtagagg aaacttgaag acattttcac tggaaaggaa 53220
    gagaggaaga ggagggagat gtctgtaagg acgagcaaac cgggtgacag ctgatttcct 53280
    catattgaag taatgagtcc tagttataat aaattcctaa taaaaaccca gtttatccct 53340
    gcaataaact tgtctttttt ttttaaatat actgcttgat tctgtttgct aatattttat 53400
    ttacaggctt tgcattgata tgcaaaaatg agatgggcaa taattttctt tttgaatgtc 53460
    taatgttgtt tggtttcaga atcaatgtta tgctcacatc ataaaaaatt tggaaccgag 53520
    gcaggaggag tgcttgaggc cagaagttcg agaccagtct aggaaacaca gtgagacccc 53580
    cccatctcta caaaaaaaaa aaaagaaaaa aaaatgggca tgtttgcttt ttccttttac 53640
    tctgaacaat ttaaggagca ttaaaattat ctattctttg aggtttgatc atttcccagt 53700
    taaaaatgtt cctcccagcc tgatgctttc tttggggagg gtaaatcttt taaggctaga 53760
    aaagtttctt ctgtggcaat tttattattt acattttaaa aattattcta gagttaattt 53820
    tgataaagca tgtatttctt aaaacaaatt atcctttttt tccagatgtt caagtgtatt 53880
    tgcataaagt tgaggaaagt agtcttttgt gaatctttta acttctccca aatatcttat 53940
    tttgtgtatt tttgcttctt tattttgtta acttttaaaa gtgtattttt ttttcaaaga 54000
    atcagctctt aggtttatgt ttttggttat actggagctt ttttcttctt ctttttaaaa 54060
    tattttttct cctttatttt ttagacgtat tttgatctaa cgtaatcgga agaaggtaaa 54120
    ttagaatctt ttgttactat tgtgttttta tttctcctta tttctctgaa gtcctgcttt 54180
    ataaatagta ccatgttatt tgtgcataaa tattcatttg tcttatattc ttgggaattt 54240
    tcccacttca tcataaaatg accttccttg tctcatttaa tgtgttcaaa ctttgccctg 54300
    aatttaactt tgtctgatat tttaccatcc tgctgaattt tgtttgttac cccaaacaac 54360
    ctttgctgtt ttcgtctttt ctgaaccctt tattttaggt aatcccttga attagagcac 54420
    taagttttgc tttgtgatta aatctgaaaa tctttatctt gccatagatg agttgagccc 54480
    tattcatgtg acagctatat tatgctgttt catagccctt ttggtccttt tttcactctt 54540
    gcattgcata ttttgtgttt attgtgtttt gtgtttcttc tgataatttg gaaggtttgt 54600
    atttttattc agggagttgc cttataatca tactccgcaa tacacatcgt cctcagtttc 54660
    ttcagactgt ctgttaactc cctattctga ataaaaatga cattgtaatt tccctctttt 54720
    ttctttaccc cttttcttct cctcacctaa tgtaaatgat tttatccttc tttagtattt 54780
    gcttttttaa ttaactacat ttataaatat ctttatcact tgatttttaa atcagctttg 54840
    aatgagatat ttggattcct agatataaaa gatgttaatt ataccatttc cacgttagta 54900
    ggtttataaa atcatacatt ctgctgtgta accataatcc cacgtttgtt ttagttccac 54960
    tcctacagtt aaaagattca gaagtattat taacagttat tttgccatag ttttttcccc 55020
    aacccatttt gtggtaagtt atgatcctgc tttagtttct taagaataat ttatagagca 55080
    gagtgtggtg gctcacgttt gtaatcccag cactttggga gacaagaggt agaaggatcg 55140
    cttgaagcca gcagttcaag accaccctga gcaacatagt gagaccttgt ctctacaaaa 55200
    aattttaaaa tttagccaga cgtagtggcg tgtgcctata gtcccagcta ctcaggaggc 55260
    tgaggcaaga ggattgctag agcccagaag tttgaggctg cagtgacctc tgattgtgcc 55320
    actgcacccc agtctgggca agaaagtgag aacctatctc tttaaaataa caataataac 55380
    ttatgaaaat tatattccct gagtttttca tgtttaaaaa tatttgttgc ctttatcctg 55440
    taaaagtttg agtataaatt cttgggttat actttattta ttgaagaatg tataagtatt 55500
    gtcttctaga attgagtgtt gctgtaatga aaccagaagt cagcctggtt tatttttcct 55560
    cagaaatgag gtaattgccg gccggacacc gtggctcatg cctgtaatcc caacactttg 55620
    ggaggccgag acaggtggat cacgaggtca ggagattgag accatcctgg ctaacatggt 55680
    gaaaccccgg ctctactaaa agtacaaaaa gttagctggg catggtggtg gacgcctgta 55740
    atcccagcta cccgggaggc tgaggcagga gaatggcgtg aacctgggag gaggagcttg 55800
    cagagagctg agatcgcgcc actgcactcc agcctgggcg acagagtgag actccgtctc 55860
    aaaaaaacaa aaaaaaaaca aagaagtgaa gtaattgcca tgatgctcca agaattatct 55920
    ctttgtctat gaaatccaga aatctcactg ttatacattt tggaattatt attctgggcc 55980
    aatatttcct gggacacaat agattgactc tatagattta attttttttt tttttttgag 56040
    acagagtctc actgcaatct cagcttactg caacctctgc ctcacgggtt caagcaattc 56100
    tcctgcctca gcctcccaag tagctgggac tacaggcgcg tggcaccatg cctggctaat 56160
    ttttgtcttt ttagtagaga cagggtttca ccatgttggc caggctggtc ttgaacgcct 56220
    aacctcaagt gatccacctg cctcagcctc ccaaagtgct gggattacag gcgtgagcca 56280
    ccatgcccag cctcaattcc tctttctatc tggtaatttt tctgaagttg aaaacatttg 56340
    ttctaatacg ttatttcagt gttcttctaa gatgtgtaaa gcaccctatt cccaggtcag 56400
    cccccatctt gctagtgagc tcggctggtt cttcacaaga gctctggttt tctcctgctt 56460
    aatctcaagt acctctgtca gcctccacct ggtttatgat ttggagtttt ttggtttttg 56520
    ttttttgttt ttgacagagt cttactctgt cacccaggct ggagagcagt ggcataatct 56580
    cagctcactg caacctctgt ctcccaggtt tgagcgattc tcctgcctca gcctactgag 56640
    tagctgggat tacaggcgcg tgccaccaca cccggctaat ttttgtattt ttagtagaga 56700
    tggggtttca ccatgttggc cagggtggtc ttgaactcct gacctcaggt aatccacctg 56760
    cctcagcctc ccaaagtgct gagattacag gcgtgagcca ccgcgcctgg catggtttgg 56820
    agttttaatc tgtagtttta ataaagatag tgcttatgtt tgtgtttctt atatttcttg 56880
    gtactcttgg gtaatttgta agatccccat atctacacaa gaagtccatt ttcaattctt 56940
    ttcttcagac tgtttatttt attttatttt attttatttt tatgtttgag atggagtctc 57000
    gctgtgtcac ttctggaggc tggagtgcag tggcgcgatc tcaggtcact gcaacctccg 57060
    tctcccgggt tcaagcaatt ctcctgcctc agcctcccga gtagctggga ttacaggcac 57120
    ctgccacttt ttaatttttt tagagacaga gtctcgcttt gttgaccagg ctggagtgcg 57180
    gtggtgcaat catggctgac tataacctcc aaatcctggg ctcaagtgat cctcctgcct 57240
    cagcctcctg agtagctggg actacaggca catgccacca tgcccagtta attttaattt 57300
    ttttgtagag acagggtctc catatgttgc ccaggctggc ctcctactcc tggcctcaag 57360
    taatcctcct acctcagcct cccaaattac taggattata agcatgagcc accatgccca 57420
    gccttgttct actactttaa tttcatatgt taggtgacca tgtaattgat catccaaacc 57480
    aggatactgt aagaatgaaa gaggctgaca gtagtatgat gctgggacta gcattgtgca 57540
    ctgagattat ttctgggaaa gcaggagata cggtcaccct acttatagtg tgcttgtctt 57600
    tggattgttg aatttggagt ttctatttgc aggcttattt caactgggca gccttgatcc 57660
    gccctgccca gcaatgctac cgttctctcc accgggtctc tgggacccct tcagtcacta 57720
    tacttagctc agttccccac cctcccactc cctaaaagcg taaccaggaa tcctgcctca 57780
    ggtctactgc cgtcttccgt gggctgtttc agttcctatt acccagagtc aaactcccag 57840
    cattccctac ctgattccag acttggagtc cagagcttta acctcttcag gccaactccc 57900
    cactttgcat ttctgtccct atatcttagt ccatggagat acatttcatg tctttgagtc 57960
    tacttacaaa gtaaattttg ctgtttttta attttttttt tgagatggag tcttgccctg 58020
    tcacccaggc tgtggtgcaa tgacgccatc tcggctcact gcaacctccg cctcctgggt 58080
    tcaagcgatt catctgcctc agcctcccaa gtagctgtga ttacagacag gcaccaccac 58140
    gcccagctaa ttttttttat cttttagtag agacagggtt tcaccatgtt ggccaggctg 58200
    gtcttgaatt cctgacctcg tgatctgccc atctcggcct cccaaagtgc tgagattaca 58260
    ggcgtgagcc actgtgccca gccaattttg ctttttttat atttcattgc tatatgttta 58320
    gaggataagt ttacagtgct atatgcattc ccaaatatta gaccaaaaaa atctccaaaa 58380
    aattagaaag aaaatccaaa aaatctcaaa aaataccaaa aagcaacaat ctcacagacc 58440
    atactcactg acccccaata aaataaaatt agaaattaac cacaacttaa caaaataaag 58500
    tactcaagtc agagaggaaa gaggaaataa acatcaaaat tacaaagtct aggcggtggc 58560
    tcacgcctgt aatcccagca ctttgggagg ccaaggcggg cagatcacaa ggtcaggaat 58620
    tcgagaccag cctggccaat atggtgaaac cccgtttcca ctaaaaatac aaaaattagc 58680
    caggcatagt gatgtgtgcc tgtaatccag ccacttggga ggctgaggca ggagaatcac 58740
    tgaacccagg gagacgaaga ttgcagtgag ccaaaatcgt gccactgcac ttcggcctgg 58800
    gtgacaaagc gagactccat ctcaaaaaaa aaaaaattac aaactcttta gatagaaatt 58860
    ttggtgtttt tttttgagac ggagtctcac tctgtcgcag aggctggagt gcagtgggac 58920
    tatgtcagct caccgcaacc tccatctcct ggattcaagc aattctcctg tctcagcctc 58980
    ccaagtagct aggattacag gcgcccacca ccagacccag ctagttttta tatttttagt 59040
    agagatggtg tttcaccatg ttggccaggc tggtctcaaa ctcctgacct caagtgatcc 59100
    acctgcttca gcctcccaaa gtgctcagat tacaggcgtg agccaccgca ccccacctag 59160
    atagaaattt caacatgagg ccgggcacaa tggctcacgc ctgtaatctc agcacttcag 59220
    gaggctgagg cgtgggagga tcacttgggc ccaggagttc aggaccagca tgggtgacag 59280
    agacagaccc tgtctctatt tatttgaaaa aaaaaaaaaa aaagagagag agaaagaaat 59340
    ttcaacatga aaagtatctc tcaaaccctt cgagatgttg gcaaaaagcg actcaaagga 59400
    aaatgtatta ctgtgtgtga atttgcttga aaataagaaa gaggccgggt gtggtggcta 59460
    acacctgtaa tcccaacact ctgggagtcc gaatcaagtg gatcatgagg tcaggagatc 59520
    gagaccatcc tggctaacat ggtgaaaccc tgtctctact aaaaatacaa aaaattagct 59580
    aggcgcggtg gctcatgcct gtaatcccag cactttggga ggctgaggca ggtggatcac 59640
    ctgaggtcag gggtttgaga ccagcctggc ctacatggtg aaacctcgtc tcttctacaa 59700
    atacaaaaat tagctgggcg tggtggtggg tgcctgtaat cccagctact cagaggctga 59760
    ggcaggagaa tcgcttgaac ccgggaggcg gaggttgcgg tgagccgaga tcgcaccact 59820
    acactccagc ctgggcaaca gcctgggtga cacagtgaga ctccatctca aaaaatacaa 59880
    aaaattagct gggtgtggtg gcctgcgcct gtagtcccag ctacccggga ggctgaggca 59940
    ggagaatgga gtgaacctgg gaggaggagc ttgcagtgag ccgagatccc accactgcac 60000
    tccagcctgg gcgacagagc aagactcttg tctcaaaaaa aagaaaaaaa aaggaaaaaa 60060
    gaaccctgat aataaagaaa ccaaatgttc aactctcaaa gctcggacac tttaaagaaa 60120
    taattaataa aggcagaagt taaagggagg atgataaagc aatttttttt gttggttttt 60180
    ttgagatgga gtcttgctct gtcacccagg ctggagtgca gtgatgcgat cttggctcac 60240
    tgcaacctct gcctcccggg ttcaagcaat tctcctgcct cagcctcctg agtagctggt 60300
    actacaggtg cgcgccacct ggcccagcta atttttgtat ttttattaga gacggggttt 60360
    caccatattt gttaggctgg tctcaaactc ctgatctcag gtaatctgcc cacctcggcc 60420
    tctcaaagtg ctgggattac aggcaggcgc caccgcgcct ggcctaaagc aaaatattgg 60480
    ttctgtgcaa aaggtcaata aaaagagcaa acgtttacaa actggagcca gcacccattc 60540
    agctcagtgt gtctggagaa aaaacaatct cgcttcagaa ttcatgatta cgcagccctt 60600
    tttgcttcct aaaaatccta ctatgttgct gttgaccatt ctctctcttt ctctctctct 60660
    tgctttctct ccagaaaagc tattcagaca ttctcctctt tcctcaaacc tccaacactt 60720
    cctcctccat ccttagcctc agctgctgac ctcacttcta atcattgaga aaccaggaga 60780
    agcatttaag agtgaacctc cgcctccccg cacgggcaaa accacccacc cacagaattg 60840
    tgccccaatt ctgcgtcctc tcctctcacc atggatggac ggtccaggct ccgagccaaa 60900
    gccaggcctc ccctggagct ctggatccac cacctgcagc ttctcaggca gggccccagc 60960
    agctcccctg ctcccttgta ccatcaatcc ctcccctcac tgggtcactc ccaacaatat 61020
    atatatttag tgatgtttct cccatgtggt aaaatcactt agcctctctc ctcccccagc 61080
    tactatccta tttgtttctt tccattctct gcaaaacttc tcaaagcatt gtgtctatgt 61140
    gctgactcca tttatcttct cccgttctct gctgagtcct tcccacagac tctcacccca 61200
    gttactccat gaaatgacct ctgcactgcc acatccaatg gtgaatgttc agttcttaat 61260
    tttattcagt ctttcagcag catttgacct ggccgatcac tccctcttct taaaaatact 61320
    tttctcagcc aggcgtgatg gctcacacct gtaatcccaa cactttggga ggccaaggcg 61380
    ggaggatcat gagagcccag gagttcaaga tcagcctggg caacatggca agaccctatc 61440
    tctacaaaaa ctaaaaagta gccagtgtga tggcatgcac ctgtagtccc atctacttag 61500
    gaggctgagg cagtaggatg acttgagcct gggaaatcaa ggctgcagtg agccatgatt 61560
    gcaccactgc actccagcct gagtgacagc gagaccctgt ctcaaaaaga caaaatagga 61620
    aacttttctc agcatattcc tctgattctc ctgctgcttc tgtctgcaca gattcagtct 61680
    cctttgccgg ttcttcctca tcctcctgat ctcttgacct tgaagtgccc cagagtacag 61740
    tctttttttt tttttttgag acgcagtctc gtctgtcacc caagctggag tgcaatggcg 61800
    aggtctcagc tcatgcaacc tctgcctcct gggttcaagc gattctcctg cctcagcctc 61860
    ccaagtagcc aggactacag gcacatgcca ccatgcccag caaattgttg tatttttagt 61920
    agagacaggg ttttactata ttggccacgc tggtctcaaa ctcctgaact cgtgaaccac 61980
    ccgcctcggc ctcccaaagt gctgagatta caggcatgag ccaccacacc cggcccagag 62040
    tacagtcttt agacggcctc tctacctata cttgctcccc tcataaactc ctcctgcctc 62100
    atggctttaa ataccatcgg tagactgatg actcccatat ttctcttttt tttttggaga 62160
    cggagtctcg ctcagtcccc caggctggag tgcagtggcg cgatctcggc tcactgcaag 62220
    ctccacctgc caagttcaca ccattctcct acctcagcct ctccagtagc tgggactaca 62280
    ggcacccgcc accacgcctg gctaattttt ttgtattttt agtagagatg gggtttcacc 62340
    atgttagcca ggatggtctc gatctcctga cctcgtgatc cgcccatctc ggcctcccaa 62400
    agtgctggga ttataggtgt gagccaccgt gcccagccga tgactcccat atttctatct 62460
    cttgctgtgt gggagttctc ctcagaactc catactcata aatccaactc tcataaatag 62520
    tatctcaaat gggcaatatg ctcaaaagtc aattcctact tttctcccta aacttgcttt 62580
    cctgcagtct ccaccatctt aatgtccaat ctaacattag gaggcaaaaa ctttgaagtc 62640
    attcttgact cttctctatt acacacccta tccaatcttt ctgcagatcc agtcgacccc 62700
    caaatccagt tagctctcat catctcccct gttaccccct ggtccaggcc atcttcctct 62760
    ctcacctgaa tcactgcagc attctcctca ctggtctctt tggttctgtt ttcactccac 62820
    cttagcatag tctccacaga gcagtcagag ggatcctttt aaagtgtaat tcccatcctg 62880
    tccctgctct gctcaaaacc ctgtcgtgat tcccgtttta atctgtcaga ttaaaagcca 62940
    gagtctttcc agtgacctac atgatctgcc tattatcacc tcccacttct ttccccttgc 63000
    tcactccact ccagctctgc agctgtcctt tctgtttcct gaacagccca gattttgctt 63060
    ctttagaacc tttgtatttg ctgtcccctc tgtctggaat gtttttccag gaagtcacct 63120
    ggctctctcc tgcacttcct tcctgaccac catgtttaaa aatcactcaa acacacttca 63180
    ggccggacat ggtggctcac gcctgtaatc ccagcacttt gggaggccaa ggtgggtgga 63240
    tcacctgagg tcaggagttc gagaccagcc tggccaacat ggtgaaactt cgtctctact 63300
    acaaatacaa atagtagcca ggtgtagtgg cacacacctg taatctcagc tactcaggag 63360
    gctgaggcag gagaatcgct tgaacccaga aggcagagga ggtgcagtga gccaagatca 63420
    cgccacaaca ccccagcctg ggtgacagag caagacccca tctcaaaaaa aaaaaaagaa 63480
    aaaaaaatca cacaaacaca cttctcttca tattcctttt ccaagtttta tttttctcca 63540
    gaatacttta cattgtttta atggaagttc tccgtttccc cccaactaga atggatactt 63600
    cctgcaggta ggcactctag tcctcccatc caagtactaa ccaggctcaa ccctgcttag 63660
    cttctgagag caggggagat caggcctgtt cagggtggta tggcccagga attttgattc 63720
    tgttttattc attgctgttc tgttgattct cttttgttcc tcctcctagt gctgagaaca 63780
    ctacttgtac ataataagca ttcaataaat atttgttgaa tgaatgactt gttgaatgaa 63840
    ttaatctcag aaatgcagga ctggttctac attagaaaat ttttcaaggt cattctctgt 63900
    tgtcgtaaca cattaagaga ggaaaatttt gtactctaaa tcatttgata aaatacatac 63960
    tgatttctgt tttcaaaaac tcttagtggc tgggcgaggt ggctcacatc tataatccca 64020
    gcattttggg aggacgaggt gggcggatca cttgaggtca ggagtttgag accagcctgg 64080
    ccatcatggt gaaaccctat ctctactgaa aatagaaaaa ttagccgggt gtggtggcgc 64140
    atgcctgtag tcccagctac ctgggaggct gaggcaggag aatggcttga acccgggagg 64200
    cggaggttgc agtgagccaa gatcatgcca ttgcactcca gcctgggtaa cagagtgaga 64260
    ctccatctca aaagaaaact cttagtgagt ttaggaatcc aaggaagacc ctcaaactaa 64320
    atagataatc tagctaccag aagccttcag taaaccttaa cactccatgg tgaaacatta 64380
    gaaacattcc tactaaaaga caggctaaga atgcctgcaa tcttcacggc tagtccaaga 64440
    agtcaaaaag aagaaatgag cgctgattta aaaaaataaa caaacaaaaa actaccgatg 64500
    cagaggctgg cagcaaggac tgaaggactg tacagtactt gcctggagca ggcggatggc 64560
    cacacccctg cgaagcctgc tcagctggct gggggacgct ccagtgtgtg agtggcagga 64620
    tgcagggtac ttcctctgcc agggagttgc actggggaga tcctccccca ctcacacttt 64680
    ggcagctggg gctttggaat gtgacttagc ttctgtcaaa gggtcaatcc accctttgat 64740
    atatgatgca aaggcgaaca tatgatgcaa aggtgagaga acagcccaaa ttaggacttt 64800
    taccacagct gtggaggtgg acagcgacag tggtgggccc tggccagact tttcatgctc 64860
    aaaggtggtg gttgttcttc ctacttcttg tccctccagg gcttcctttg cctgtgtgct 64920
    gaacctgctt cttttaattt tttttaactt ttttaaattt ttaattgttt taattaaaac 64980
    aaattttgaa aactgtctga acctgctttt gaaccctgct atgatttgaa tgtttgtccc 65040
    ctgccaaact gattttgaaa cttaatctcc aaagtggcaa tattgagatg gggctttaag 65100
    cagtgactgg atcatgagag ctctgacctc atgagtggat taatggatta atgagttgtc 65160
    atgggagtgg catcagtggc tttataagag gaagaattaa gacctgagct agcatggtcg 65220
    ccccttcacc atttgatatc ttacactgcc taggggctct gcagagagtc cccaccaaca 65280
    agaaggctct caccagatac agctcctcaa ccttgtactt ctcagcctct gtaactgtaa 65340
    gaaataaatg ccttttcttt atgaattacc cagtttcaga tattctgtta taaacaatag 65400
    aaaacgaact aaggcaaact ctcatgattc tactgccatg ccattccaat aaactccctt 65460
    tatgcttaag agagccagag ttggccaggc gtggtgactc acgcctgtaa ttccagcact 65520
    ttgggaggcc gaggcaggtg gatcacaagg tcaggagatc gagaccatcc tggctaacac 65580
    ggtgaaaccc cgtctctact aaaaatacaa aaaaattagc tgggcgtggt agtgggtgcc 65640
    tgtagtccca gctactcggg aggctgaagc aggaggagaa tggcgtggac ccaggaggcg 65700
    gagcttgcag tgagtcgaga tcgtgccact gcactccagc ctgggtgaca gaatgagact 65760
    ccgtctcaaa aaaaaagaga gccagagttt atttctgttg cttgcaacca agaaatctgg 65820
    ctggtgcact gaagtttcca taaataatag caatttaaag actctttcca agccaggcaa 65880
    tgcctagcct tgtgtagtcc ttgtggtaat acattcattc attcatttgt tcaaccaact 65940
    gtgctccaga gactaagaat acaaaaatgg gggccgggtg tggtggctca cacctataat 66000
    cctagcactt tgggaggccg aggcaggtag atcacctgag gtcaggagtt cgagaccaac 66060
    ctggccaaaa tggtgaaacc cctactctac taaaaataca aaaaattagc tgggggtggt 66120
    ggcggacacc tgtaatccca gctactcgtg agactgaggc aggagaatca cttgaacccg 66180
    ggaggcagag gttgcagtga gccgagatcg caccactgca ctccagcctg ggcaacaaga 66240
    gcgaaactcc acctcgaaaa aaaaaaaaaa aaaaaaagag ggccggggct gggcgcagtg 66300
    gctcacgcct gtaatcccag cactctggga ggccaaggca ggagaattac gaggtcagca 66360
    gatcgagacc agcctgacca acatggtgaa accccatctc tactaaaaat acaaaaatta 66420
    tccgggcgtg gtggcgcaca cctctagtcc cagctacttg ggaggctgag gcaggagaat 66480
    cgcttgaacc cgggaggcag aggttgcagt gagccgaaat catgccactg cactccagcc 66540
    tgggtgacag agtgagactc cgtctcaaaa aaaaaataaa aaaaaaaaaa gaattcaaaa 66600
    attgtagagt tatagtgtgc ttctagttta gttgagagga catctgtcct tcaaggaagg 66660
    ctagaatcta taccctgagt ccttactgaa atcaatccag cagtcaaaac atgggaccaa 66720
    cgatcacagc agtaagatag gaagagcacc tttgtacatt tagctcatgt tgagataagc 66780
    cactgacaga gctgaaggaa gctcacagtt ctgggttcca tcctttggca tttaaaaaga 66840
    aaagtgctaa gaaaattcgg ttggtcacgg tggctcacgc ctgtaatccc aacactttga 66900
    gaggccaagg caggcagatc acgaggtcag gagttcgaaa ccagcctggc caacatggtg 66960
    aaaccccgtc tctactaaaa acagaaaaat tagccgggca tggtggcgca tgcctataat 67020
    cccagctact caggaggctg aggcaggaga attgcttgaa cccgggaggg ggaggttgca 67080
    gcgagtgaga gcaggccact gcactccagc ctgggagaca gagcaagact ctgtctcaaa 67140
    aaaaaaaaag aaaaaaagaa agaaaggaaa aaaagaaaga aaaaaaaaga aaaaagaaaa 67200
    ttcaggccag gccaggcctg gtggctcaca cctgtaatcc caacactttg ggaggctgaa 67260
    gcgagacggt gccttagccc aggagtttga gaccagcctg agcaacatag cgagaccctg 67320
    tctctataaa aaaaaatttt tttttggcca gacgcagtgg ctcacgcctg taatcccagc 67380
    actttgggag gccgaggcag gtggatcacg aggtcaggag atggagacca tcctggctaa 67440
    cacggtgaaa ccccatctct actaaaaaat acaaaaaatt aaccgggcgt ggtggcgggc 67500
    gcctgtagtc ccagctactc gggaggctga ggcaggagaa tggcgtgaac ccgggaggcg 67560
    gagcttgcag tgagccgaga ttgcgccact gcactccaga ctgggagaga gtgagactcc 67620
    gtctcaaaaa aaaaaaaaaa aaaaaaaaat taattgtcag gtgtgctggc atgcagctgt 67680
    agtcctagct actcgggagg ctgaggtaag aagatcgctt gagcccagga gttcaaggct 67740
    gcagtaatag tgcctctcac tctaccctgg gtgacaatga gaccctctct caaaaagaaa 67800
    gaaaaaaggg aaagaagaaa agaaagaaag aaagagaaga aaggaaggaa gaaagaaaga 67860
    aaaagaaaag gaaggaagga agaagaaaaa aaaagaaaga aagaaaagag agagaagttc 67920
    aaagaccaaa gggtcaggat cccaaaatag tttttatgtt ttatttattt atttacttat 67980
    ttatttttga gacagtatgg ctctgtcgcc caggctggag tgcagtgatg cgattgcggc 68040
    tcactgcagc ctccaaactg ggctcaggtg gccctcccac ctcagcctcc cgagtagctg 68100
    ggaccacagg cgcgtgccac catgcccagc taatttttta attctttgta gagatgaggt 68160
    ctctatatgc tgcccaggct ggtctcgagc tcctgggctt aagccatcca cccgcctggg 68220
    cctcccaaag tgctgggatt acagaagtga gccaccgcgc ctaatcgggt ggtttgtttg 68280
    tttattgacg gggtctcgct gctgcccagg ctggagtgcc agtggctgtt cacaggtgca 68340
    gtcctggagc attgcatcag ctcttgggct ctagcgatcc tccagagtag ctgcagctgg 68400
    gattccaggc gcgccaccgc gcggggctca gaatgggttt ttatattgag ggttatgctg 68460
    ccacctagag gatatatgta gtaccgaact gtgtgcgcag ggaggctgag gttgcagtga 68520
    gccaagatga tgccagggca ctccagcgtg ggtgacagag caagatttca tctcaaaaaa 68580
    aaaaaaaaaa aaaaaaaaaa aagaattgaa agtaaggtct tgaagagata tttgtgcctg 68640
    tatggtcata gcagtattaa ctttgaccca ctagctaaaa cacaaaagca acatgtgtct 68700
    gtcagcaggt gaacggataa acaaaatgtg gtatatatgt acaattgaat attattcagc 68760
    ctttaaaaag gaataaaagg ctggatgcgg gggctcacgc ctgtaatcct aacactttgg 68820
    gagactgagg tgggtggatc acccgaggtt aggagtttga gaacagcctg gccaacatgg 68880
    tgaaacttca tctctactaa aaatactaaa attagccggg catggtggca cttgtctgta 68940
    atccaagcta ctggggaggc taaggcagga gaattgcttg aactcaggag ccggaggttg 69000
    cagtgagcta agatggcacc actgcactcc agcctgggca acagagtgag actccatctc 69060
    aaaacaaaca aacaaaaaat tattatttcc aaagaaacaa gaccctgggt ccatttccca 69120
    gcccacacct gatgttgact cacaacacac agcctggttt gctatgagcc tgcttcattt 69180
    aattgtcacc ttaacttcac atcaccctca agtcctggaa taactctttg ctgacctttg 69240
    tgtgctgagc catctccatg tcgctcaacg tgcagtccct ctcactgcac tgagtcaata 69300
    gccagacgtg gtctgactgc agggtcatcc ttggtggctt aggctgactc gggcatagca 69360
    gggtgctctg agacctcacc gcatataggc tttgccccca ataaactcta tataatattc 69420
    atattatgtg gtctgggtgt gtgtagcttt gcactgtctt ctcgtgacag tgccctcaac 69480
    ctctttccca ggatttcctc ctctacctcc tcaagtccca ctgctctgca aagaccaaaa 69540
    gctgcagagt cccagctccc tcctttacac cccacgacgc agcctcctct ctcagaaccc 69600
    tttaaacaga gtcttttact gcagatccca agaacagcca cacccctctc tcccacccac 69660
    tccagacaca cccaggtaat tatagcaccc agggtaacta tgtagatgga gtccctggaa 69720
    catgtggata gtgccccctg ggagtatgca aaagcaacat tgctggcacc tgcagagaac 69780
    agggtgacat ccaggaatca gagcatgggc ctctgggagg tagggatgtg gccaggcagg 69840
    ctgccaaaaa ttggtagagc aaggccacag gatctttctg accttccttc caaacagagg 69900
    ctcctgtact ggtgatccct gtgttgattg accactccct tcctgggggt cgtggtctct 69960
    gtcccagttg cccggacttc tgtgagtgtc ctactgaggt ccttttcatg agaagcatgc 70020
    tgtccttcca cctgctggga gcaagagtga caacttcaat actataatag cagtggcata 70080
    cagagaagaa gaaagatgaa gtggcaagaa aaacaggctt ccaagcagga gtttttctat 70140
    aaaaacaaaa acgtttacaa gcaaactttt tataaagggc tagatagtaa atattttagg 70200
    ctttgagagc cacatagact tgtttgcagg gactcaatgt cgctattgta gtttgaaagc 70260
    agccatcagg gttatgtaaa tgagtgagtc tgattttgtt tcagcaaaat tttatttacc 70320
    aaaacagaca atgagtgggc tggatttggc ccatgatcct tagtttgcca actcctgctt 70380
    tgggctcacc cagatctgat tttgaattct ggctctgcta ctggttagct gcaggagctt 70440
    ggaaggctct ctgagcctgt ttcctcatct gtaaaattaa agcaataatt tctaacactc 70500
    aagagtgtta cctcacgcct gtaatcccag cactttggag gctgaggcag gcggatcacc 70560
    tgaggtcaga agttcaagac cagcgtggcc aacgtggcaa aaccctgtct ctactaaaaa 70620
    atacaaaaag tagccgggca tggtggcgcg catctgtaat cccagctact tgggaggctg 70680
    aggcagggat actgctagaa cctgggaggt ggagcgtgca gtgagtggag atcacacctc 70740
    cacactccag cctggccgac agagcgagac tccatctcaa aaaaaaaaaa aaaaagagtg 70800
    ttagaaggtt ttgagataat gaataaaaga tgccttgtgt atactaagta ttcaacaact 70860
    gatagctgca ttggtctaat tataacagtt tagaagcgat tgagtcaaca aatgctggat 70920
    ttgtcaggga ggacttccta tcaggaggta gatcttgggc tgagtcctga agcaaagata 70980
    ggcattggat agaggagttg agagaacacc ctaggactgt tattattatt attcgacacg 71040
    gagtctcttg ctctgtcacc caggctggag tgcagtggcg cgatctcggc tcactgcaac 71100
    ctctgcctcc caggttcaag cgattctcct gcctcctaag tagctgagac tacaggtgtg 71160
    tgccaccaca cccggctaat ttttatattt ttagtagaga cagagtttca ccatgttggc 71220
    catgctggtc tcgaactcct gacttcaggt gatccacccg cctcagcctc ccaaagtgct 71280
    ggaataacag atgtgagcca ccgcacccag cccagaacca tttttcaatc cttggctctg 71340
    ccttttatta gctgcaagat ctcaggcaat ttatttaacc tctccaaaga ctcattttct 71400
    cattcacaaa atgaggcaaa taataatatc tactatccca ggttgtcatg agaattaaat 71460
    gcaacatgac atttaatgaa atgagaagtc ccttggacat taactggcta aagtatgtgc 71520
    tcgacaagga tatcatttta ggtggatact tagcatctca gaactgatgc tcacaatgga 71580
    atatcattga aacgcattaa aattcatttt aaatgattgt aggtagtgag gcaattgaaa 71640
    gaagaagaca agaggactga ttataatgct tcaggctcac tagtctcctt ttaggaggga 71700
    aaaacaattt caagttaaat tttaggctct agatttttac ccctgctgct cattagaatc 71760
    acccagattg atgaaatcag agcccatctg aggctgtgtt tttcatctcc agaatgagag 71820
    ctgttgtggg gattaagttt ttgaaaaagt acatctaaca ggtgatcgaa aatgatagtg 71880
    atattattgc agtgatggtc attattgttg ttattattat actgaaagag gcttcagttt 71940
    tctgatccat aaagtgaggg aattgcatga gaccattgct aagattcctt ctagctctgt 72000
    ttttttgttt ttgtttttta gacagagtct ctgtcgccca ggctggagtg caatggcatg 72060
    atcttggctc actgcaacct ccgcctcccg ggttcaaatg atcctcctgt ctcagcctcc 72120
    gaagtagctg ggactacagg cacacaccac catgcccagc taacttttat atttttaata 72180
    gaggtggggt ttcaccatat tggtcaggct ggtctcaaac tcctgacctc aggtgatcca 72240
    cccgcctcgg cctcccaaca tgctgggatt acaggcatga gccactgtgc ccaacccctt 72300
    ctagctttct tgatcactga ttctagggtt ctctgctgaa atatatttga gacatcctgg 72360
    ataaaagatc atgcaagagc tcccaatatg gtattaataa ttgattctgg aggcttagct 72420
    actcctgatg gattagacat gactcaactg cctctcttat gtgtacaaca caacaacaca 72480
    accaagaaag gttattctgg cattccattt attcagttta tttacagccc ttacttccag 72540
    cagcacgtta aagatatggc cagggccggg tgcagtggct caagtctgta atcccaggac 72600
    tttgggaggc caaggtgggc ggatcacaag gtcaggagtt tgagaatctg gcaattcttc 72660
    agacttagaa gcaaccagct cgataacaca gtcttgtgtg ggctctccct ctgtccctcc 72720
    ctcgcttccc tcatttctca tccctgcccc tgagactgtg caccttcaca tagccctgcc 72780
    atgagacctt catctcaggc tttgctttct ggggtaactg aggctaaaca ctgagtggcc 72840
    ctaaaagagg attgggattt ggaagttaga ttattcacca gagaacagac tttgctgatg 72900
    atcaggccca ggttgtaatt gttgaaaaaa agagaggatg catagtctta tctcatctcc 72960
    tagtcaaagt caacaccatg ataaataaga gtcaaatcct gagatgtgaa ttggggacat 73020
    ttgagtggtt aaccctgaga agcttgcacc ttcagacccc tcaatacccc tgctccccag 73080
    agaaggctgg acattgacct cagcacaggc aggagccctg caagatgcca tttgtcctac 73140
    taaagatgga cccctccact ctgtttctag gtaaataacc aaagtcaagt ctccacacag 73200
    cctgagcaag aaagtcagag cctgctacag gagaaaatac cacactggcc aaaggattca 73260
    ctagccctgg ccactgtgtg tgggaggaac cagggaatca tgtgtgggag tcaatgttga 73320
    agctgttgga ctgggggtgg ggtggaatat aagcctggcc ctggggagtt tttcccgttt 73380
    gagggccttt acccacaact caagatccag tgctatagca ggagatccca gagctagtcc 73440
    taacagatgg tcaggattga acttggccta gagtaaaatg aggaggatag tgccagaact 73500
    ttctcaacat actattgagg aagaggtcag aaggcttaag gaggtagtgt aactggaaag 73560
    gggtcctgat ccagacccca ggagagggtt cttggacctt gcataagaaa gagttcgaga 73620
    cgagtccacc cagtaaagtg aaagcaattt tattaaagaa gaaacagaaa aatggctact 73680
    ccatagagca gcgacatggg ctgcttaact gagtgttctt atgattattt cttgattcta 73740
    tgctaaacaa agggtggatt atttgtgagg tttccaggaa aggggcaggg atttcccaga 73800
    actgatggat ccccccactt ttagaccata tagagtaact tcctgacgtt gccatggcgt 73860
    ttgtaaactg tcatggccct ggagggaatg tcttttagca tgttaatgta ttataatgtg 73920
    tataatgagc agtgaggacg gccagaggtc gctttcatca ccatcttggt tttggtgggt 73980
    tttggccggc ttctttatca catcctgttt tatgagcagg gtctttatga cctataactt 74040
    ctcctgccga cctcctatct cctcctgtga ctaagaatgc agcctagcag gtctcagcct 74100
    cattttacca tggagtcgct ctgattccaa tgcctctgac agcaggaatg ttggaattga 74160
    attactatgc aagacctgag aagccattgg aggacacagc cttcattagg acactggcat 74220
    ctgtgacagg ctgggtggtg gtaattgtct gttggccagt gtggactgtg ggagatgcta 74280
    ctactgtaag atatgacaag gtttctcttc aaacaggctg atccgcttct tattctctaa 74340
    ttccaagtac caccccccgc ctttcttctc cttttccttc tttctgattt tactacatgc 74400
    ccaggcatgc tacggcccca gctcacattc ctttccttat ttaaaaatgg actggggctg 74460
    ggcgcggtgg ctcatgcctg taatcccagc actttgggag gccgaggcgg gcggatcatg 74520
    aggtcaggag atcgagacca tcctggctaa cacggtgaaa ccccgtctct actaaaaatg 74580
    caaaaacatt agccaggcgt ggttgcaggt gcctgcagtc ccagcggctc aggaggctga 74640
    ggcaggagaa tggcgtgaac ctgggaggtg gaggttgcaa tgagccgaga ttgtgccact 74700
    gcactccagc ctgggtgaca gagcgagact ccgtctcaaa aaaaaaaaaa aaaaaaaaaa 74760
    tagctgggca tggtggcgcg tgcctgtaat accagctact ctggaggctg aggcaagaga 74820
    atcgcttgaa cccagtaggc ggaagttgca gtgagccgag atcttgacac tgcactccag 74880
    cctggtgaca gagtgagact ctgtctcaaa aaaaaaaaaa agaaaaaaaa agacagaaag 74940
    aaagagcaca gacagagtca caggtatttg cagtaggaag ctgtcaggtt agagtgcacg 75000
    gaaatagaaa gtatatttta cacttacagc acatcttcgt ttgattagcc acatttaaaa 75060
    tactgaatag caacgtgtgg ctatttagta ttcactaaaa tcttggacag tgcaagtcta 75120
    aagaatcctt gatccgtccg gcatggtggc tcacgccttt aatcccagca ctttgggagg 75180
    ccaaggtgga aggatcactt aaggtcagga gttcgagacc agcctggcca acatggtgaa 75240
    acctcgtctc tactaataat acaaaaaaaa ttagccgggc atggtggtgc atgcctgtaa 75300
    tcccaggtac ttgggaggct gaggcaggag aatagcttga atccaggagg cgctgcagtg 75360
    agccgagatc atgccatgcc actactgcac tccagcctgg gcaacagagt gagactgtct 75420
    caaaaaaaaa aaaaaaattg ttgggcgtgg tggctcacgc ctgtaatccc agcactttgg 75480
    gaggctgagg ggggtggatc acctgggttc tggagttcga gaccagcctg gccaacatgg 75540
    tgaaacccca tctctactaa aaatacaaaa attagctggg cgtggtggtg ggcacctgaa 75600
    atctcagcta ctcaggaggc tgaggcagga gaatttcttg aacccaggag gcagaggttg 75660
    cagtgagcca agatcgcgcc tctgcactcc atcctgggtg gcagagcaag actatgtctc 75720
    aaaaaaaaaa aaaaaaatac ttgattgtct ggacattctg cagaacatca tatggagaca 75780
    ctatgttgac gacatcatgc tgattgtaag caagaaatgg caagtgttcc agaaacacag 75840
    tcaagacaca tacatgccag aaggtgagat ataaactcta ctaagattca gtggcctgcc 75900
    acactggtga catttttaaa cctgctagat gtttgtgtag aaaaggattt aaccttgccc 75960
    aaagaggggt ctggcctttg tccccagcta ctggacataa tctctttaaa ctcttgaaat 76020
    atcattcctg atagaagtat ttttgttttg actaggggcc ttgggccagc cagatagcaa 76080
    caatgtgatc tgggttgggg gctttggatc aggtggcatc agtgtgacct cctgagtggc 76140
    tagagactag aatcaaccac atgggcagac aacccagctt acatgatgga attccaataa 76200
    agactttgga cacaagggct tgggtaagct ttcctggttg gcaatgctct atactgggaa 76260
    acccattctg actccatagg gagaggacaa ctggatattc tcatttggta cctccctggg 76320
    ctttgcccta tgcatttttc ccttgtctga ttattattat tattatgaga tggaatctcg 76380
    ctctgtcacc caggctggag tgcagtggaa tgatctcaac tcactgcaac ctctgcctcc 76440
    ccggttcaag cgattttcct gtctcggcct cccgagtagc tgggactaca gatgcatacc 76500
    accacacccg gctaattttt ttgtattttt agtagagacg gggtttcacg ttagccagga 76560
    tggtctcgat ctcctgacct catgttccgc ctgcctcggc ctctcaaagt gctaggaata 76620
    catgtgtgag ccaccgcgcc cagccccctt ggctgattat taaagtgtat ccttgagctg 76680
    tagtaaatta taaccgtgaa tataacagct tttagtgagt tttgtgagca cttctagcaa 76740
    attatcaaac ctaaggatag ccttggggac ccctgaactt gcagttggtg tcagaaataa 76800
    gggtgctcat gtgtgtacca tgccctctaa ttttgtagtt aattaacttt cacaacttta 76860
    ttattaccgc ttacactcaa tgtttattca catttatcca cataccactt attctagtgc 76920
    cttgcatcaa agactttcta tctcatgtac tttattctgc ttgaagtaaa tcctttagga 76980
    tattcttttt tttttttaaa ctttgcacat acatactttt attttttatt tatttttaat 77040
    tttgttattt ttgtgggtac gtagtagata tatgtattta tggagtacat gagatgtttt 77100
    gatacaggca tgcaatgtga aataagcaca tcatggagaa tggggtatcc atcctctcaa 77160
    gcaatttatc cttcaagtta caaacaatcc aattacactc tttaagttat tttaaaatgt 77220
    acatttaatt ttgtattgac tagagtcact ctgttgtgct atcaaatata attttttttt 77280
    tttttgagac agagtctcac tcagtggccc agactgaaag tgcagtggca caagctcggc 77340
    tcacttcaat ctctgcctcc ctggttcaag cgaatctcct gcctcagcct cccacatagc 77400
    tgggattaca ggcacacacc accatgccca gctaattttt atattttttt agtagagacg 77460
    ggttttcgcc atgttggcca ggctggtctt gaactcctgg cctcaaatga tctgaccacc 77520
    tcagcctccc aaagtgctag gattacaggc atgagccacc acacctggcc aaaatagaat 77580
    attctttagt gaggtctgct ggtgacaatt tttttctttt ttttgagact gagtctcgct 77640
    gttgtcagct tgggctggag tgcaatagca cgatctcagc tcactgcaac ctccacctcc 77700
    cggattccag caattctcct gcctcagcct cccaagtagc tgagagatta caggcaccca 77760
    ccaccacacg cggctaattt ttgtattttt agtagaaatg ggggttcacc gtgttggcca 77820
    ggctggtctc gaactcctga cctcaggtga tccacccacc ttggcctccc aaagtgctgg 77880
    gattacaagc atgagccacc acgcacagcc aattttttcc gtttttgtct gaaatcttat 77940
    tttgtgtcat ctttgaaata tatttttgat ggatataaaa ttgttggttg atagttatta 78000
    tcattattat tattattttg agacagggtc tcactctgtt gcctatgctg gggtgtagta 78060
    atgtgatctc ggttcactgc agacttgacc tcctagggct caggtgatct tcccacctca 78120
    gcctccctag tagctgggac tacagatgca tgccaccata cccaactaat ttttctattt 78180
    tttgtagaga tgaggctttg ccacatttcc caggctggtc tctaactcct gagctctagc 78240
    aatccaccca ccttggcctt acaaagtgct gggccatgac tagccagcag ttacttttta 78300
    tagcatattg aatatttaat atgaatcttc tggcatccac tgtaactgtt taaaaaatca 78360
    gctgtttact tggcactctt tttttttttt ttttttttga gacagagtct tgccctgtcg 78420
    cccaggctgg agtgcagtgg cgtgatcttg gctcactgca agctctgcct cccgggttca 78480
    cgccattctc ctgcctcagc ctccggagta gctgggacta aaggcgcccg ccaccacgcc 78540
    cggctgattt ttttgtattt ttcgtagagt tggggtttca ccgtgttagc caggatggtc 78600
    tcgatctcct gacctcgtga tctgtccgcc tcggcctccc aaagtgctgg gattataggc 78660
    gtgagccacc gcgcccagcc tctttttttt ttttttttag acggagtctt actctgtcat 78720
    ctaggctggt gtacagtggc gtgatctcag ctcagtgcaa cctccacctc ctgcctcagc 78780
    ctgccaaata gctgggatta caggtgcgta ccatcacgcc cggctaattt ttgtattttc 78840
    agtagagatg gggtttcacc atgttagaca ggctggtctc gaactcctgg cctcaagtga 78900
    tctgcctgcc ccagcctccc aaagattaca ggcatgagcc accgcacccg gccaagtagc 78960
    actcctttga aggtaatctg cttcccctac ccctagcaat ttttaacaat ttttcttcat 79020
    ttttatttcc tgaagttttg ttattaataa tctgtgtgca gatttctttg tatttctttt 79080
    gtttgcagtt catagtgatt cttgaattag tgtgttggtt tctgttatca ccacaggaaa 79140
    attgtcagcc gttagctttt caaatatttc cttgctaaat tctctcttct cccctttcgg 79200
    tacaattgat ttgattaaaa ctaaaaccag ggccgggtgc agtgactcat gcctgtaatc 79260
    ccaacacttt gagaggctga ggcaggtgga tcacctaagc tcaggagttc aagaccagcc 79320
    tggccaatat ggtgaaaccc cgtctctact aaaaatacaa aaattaccag gcatggtggc 79380
    acacatttgt agtcaggagg ctgaggcagg agaattgctt gaatccagga ggtggaggtt 79440
    gcagtgagct gagatcccac cactgcagtc tggcctgggc gacagagtga gatgagaatc 79500
    tgtctcgaaa aaaaaagtta tgaatgtttg ataaactata tttgttagaa tgtttgttgt 79560
    agaatactat tcattgattt ttaaacaatg ttagattaaa ccattcactg gatttgtgat 79620
    aattaactta ctgattttac ctcactgatt tgttgtaatt aatacaactg gtataaaaag 79680
    actgtgacga ggccgggcat ggtggctccc gcctataatc ccagcacttt gggaggctga 79740
    ggcaggcgga tcacctgagg tcaggagttc aagaccagcc tgaccaacat ggtgaaaccc 79800
    catctttact aaaaatacaa aattagccgg tcgtggtggt gcatgcctgt aatcccagct 79860
    cttcgggagg ctgtggcagg agaatcactt gaacccggga ggtggaggtt gcagtgagcc 79920
    gatatcgcgc cattgcactc cagcctgggc aacaagagcg aaactccgtc taaaaaaaaa 79980
    aaagaaaaaa aacacataaa acaaaacaac actgtgacgg ttcccaaaaa ttaggagcat 80040
    aattaaagga actcctgata aaaattaatt ttatcttaca tgtaaactaa aatgacttta 80100
    tgaagttaat tcagaaatac aatgcagggt attagtttgc cacagctgcg tattcagcct 80160
    aatgtaatat tcttgttatt tttaaattct tcttttaact ttactcatat gtggatcatc 80220
    aaatttcaaa agattaaatg acaatactct tagcagcaag cttccctaag catataaaca 80280
    ttttaatggg tgatgattca gaaggtaccc gaagaatatg tactgccaga tatcattcac 80340
    ccccatatac ctgcccgaca gacatcccat tttgggaccc tggataaatg tgtgggtgga 80400
    gagaaagata ggagaaagtg gtataagcaa atggctttgg agtctgattg acagcgattg 80460
    aaatcctgtc tctacctctt aacagcctca tgatcctaca taagttaccc cgatcctcag 80520
    ggccacatct gtaaattggg ggttgcgatg gcagccatct cacagggtct cttttcgggg 80580
    aagggcagga attatggatt aagtgagcta gtaattgtaa agcacttaat acaaggaggg 80640
    cgcataataa gtacttcata aataatgacg gccattatca tgactgaggt gtatgcagct 80700
    gtcggggatt acggcgactt cagaatttct ggtgggcagg gctcaaaggc agcaaatcac 80760
    actggaagtc gaggtgaggc actgcttctg cacagactgc ttagctggag agaatgagga 80820
    aggcttagag gagatttaga ggaacttaga gtcctccgcc tccaactctg tgggatctgc 80880
    tcccgtgcca gagacattca ggggatttct cgcactctcc cctcccctac gtccctcccg 80940
    ccccatccaa ctaaccacac aacacataca aaatagcccc tgcgaggttc tgcacgctgg 81000
    aagggaacag gagaagggcg ctgcgctttc ttgctgatgc cctgtacttg ggcccctggt 81060
    agacacagcc acttgtcccc tcagcctgca gagaaatccc acgtagaccg cgcccgggtc 81120
    cttggcttca gccaatctcc ctttggtggg ggtgggatgc acgatccaag gttttattgg 81180
    ctacagacag cggggtgtgg tccgccaaga acacagattg gctcccgagg gcatctcgga 81240
    tccctggtgg ggcgccgctc agcctcccgg tgcaggcccg gccgaggcca ggaggaagcg 81300
    gccagaccgc gtccattcgg cgccagctca ctccggacgt ccggagcctc tgccagcgct 81360
    gcttccgtcc agtgcgcctg gacgcgctgt ccttaactgg agaaaggctt caccttgaaa 81420
    tccaggcttc atccctagtt agcgtgtgac cttgagcagt tgactttatt tttcagtgcc 81480
    tagttttcca gataccagga ctgactccaa ggactattac tcatctggag ggtttagcac 81540
    agtaccgtcg catagtaaat ttccatgtca gttttggtta cctttcatgc acttgcaaac 81600
    atgccatgct ctgaaacgaa ataggcacat cttttttttt ttttttttta aggagtcttc 81660
    ctctcgccca ggctggagtg cagtggcgcg atcttggctc actgcaacct ccacctcccg 81720
    tgttcgagat tctcctgcct cagcctcctg attagctggg actacaggca tgccacgacg 81780
    cccagttaat ttttgtattt ttagtagaga cggggtttcg ccatcttggc caggctggtc 81840
    taactcctga cctcaggtga tctgactgcc tcagcctctc aaagtgttgg gattacaggc 81900
    ataagccact gcatctggcc agaaatgaaa taagtaaatc ttttaacctg ctctaacaat 81960
    atagtgaaaa gaccatatta ttattagagc aggttaaggg atttgcctat ttcgggttct 82020
    agttatagtc ttaaacttgg acattcttgt agaaagtaaa aagtttcctc ttcaaagttc 82080
    cccttcttgt taaagaatac atcataagtg ttagaagtaa tagtttattt taaagactaa 82140
    ctttcttcaa gcctccttgc tttgtgctaa taactctttg ttaagcccta tcctatgtaa 82200
    ctgttggaca tgctcacagg cacgttccag ttcacagcct atgccccttc cttatttgga 82260
    aatgttattg cttccttaaa cctttcggta agcaacttcc tctccttctt cgttcttcct 82320
    tgcacttacc tatttagaaa gttttaggct attagcaaat cggctatcag tttaagagtg 82380
    tgaggtcccg ctccagccaa tggatgcagg acatagcagt gaggacgacc caaatgcgta 82440
    agggataaat atgtttgctt ttcctttgtt caggtgtgct ctcgacatcg ttccatctgc 82500
    gattgagcac cctttctgca gaaagtaaag attgccttgc tggagatctt ttgtctccgt 82560
    gctgactttt cttcgtggca ccgattatct atttctaaca attttggtat ttctaacatt 82620
    ctgaacaatc ttgggctagt tgtctcttct gggcctgttt ccccatccgt cacatgataa 82680
    acttcattgg tttaaaaacc ccagcgaaca tttattgagt tactattacc ttcctgccct 82740
    ccccaacccc aaccccaggg agcagttaca acctcagccg ctgagcgcac tcgccgggtg 82800
    ttaagaagca ccaaagacag ggaggcttga ttgattttgc tttgggagta gagggtcaga 82860
    agattcacag gaaaatggca tttgagcaag gatgattcac tggagctagc ttttaaatac 82920
    tggcgaggct tttatgttgc agtcccttac aaagttgagc attcgcaggg actgcactcc 82980
    gaaataagcc cgcttcccct tttcattcgc taatgatcca gggagctgct ggttccgcat 83040
    gcggcaggtt gtgccttttc ctaatcaggg ttctgcatcg cctcgaaccc gcaggccgtg 83100
    gcgggttctc ctgaggaagc agggactggg gtgcagggtg aagctgctcg tgccggccag 83160
    cgcctgtgag caaaactcaa acggaggagc aggaggggtc gagctggagc gtggcagggt 83220
    tgaccctgcc ttttagaagg gcacaatttg aagggtaccc aggggccgga agccggggac 83280
    ctaaggcccg ccccgttcca gctgctggga gggctcccgc cccagggagt tagttttgca 83340
    gagactgggt ctgcagcgct ccaccggggg ccggcgacag acgccacaaa acagctgcag 83400
    gaacggtggc tcgctccagg cacccagggc ccgggaaaga ggcgcgggta gcacgcgcgg 83460
    gtcacgtggg cgatgcgggc gtgcgcccct gcacccgcgg gagggggatg gggaaaaggg 83520
    gcggggccgg cgcttgacct cccgtgaagc ctagcgcggg gaaggaccgg aactccgggc 83580
    gggcggcttg ttgataatat ggcggctgga gctgcctggg catcccgagg aggcggtggg 83640
    gcccactccc ggaagaaggg tcccttttcg cgctagtgca gcggcccctc tggacccgga 83700
    agtccgggcc ggttgctgaa tgaggggagc cgggccctcc ccgcgccagt ccccccgcac 83760
    cctccgtccc gacccgggcc ccgccatgtc cttcttccgg cggaaaggta gctgaggggg 83820
    cgccggcggg gagtcaggcc gggcctcagg ggcggcggtg gggcaggtgg gcctgcgagg 83880
    gctttcccca aggcggcagc aaggccttca gcgagcctcg acctcggcgc agatgccccc 83940
    tgagtgcctt gctctgctcc gggactcttc tgggagggag aaggtggcct tcttgcgcga 84000
    ggtcagagga gtattgtcgc gctggttcag aagcgattgc taaagcccat agaagttcct 84060
    gcctgtttgg ttaagaacag ttcttaggtg ggggttagtt tttttgtgtt tctttgagga 84120
    ccgtggatca agatcaagga aatctcttta gaaccttatt atggaagtct gaagtttcca 84180
    aatgttgagg gttttatgtc taaaagcaac acgtgaaaaa attgttttct tcacccagtg 84240
    ctgtcttcca atttcctctt tggggggagg ggtagttact gctgttacta aaataaaatt 84300
    acttattgct aaagttcccc aacaggaaga ccactacttt tgatgacttt ggcaagtttg 84360
    ctaactactg gaaccctaac ttacaaacga actacttaca tttttgattt ccagttgtat 84420
    tacctgccca atgtttacgt agaaacagct taattttgat tctgggtaac gttgttgcac 84480
    ttcattaaaa atacatatcc gaagtgagca agtatgggtc tgtggacagc agtgattttt 84540
    cctgtcaatt cctgttgctt cagataaaat gtaccagaca gaggccgggc gcggtggctc 84600
    acgcctgtaa tcccagcact ttgggaggct tggcgggtgg atcacctgag atcgggagtt 84660
    caagaccagc ctgaccaaca tggagaaacc ccgtgtctac taaaaataca aaattagcca 84720
    gggtggtggc gcatgcctgt aatgccagct acttgggagg ctgaagcagg agaatcgctt 84780
    gaacctggga ggcggaggtt gcggtgagcc gagatagcac cattgcactc cagcctgggc 84840
    aaaaagagcg aaactccgtc tcaaaaaaaa agtaccagac agaaatgggt tttgttttct 84900
    ttttttgttt tgagacggag tttcgctctt gttgcccagg ctcgagtgca atggcgcgat 84960
    ctcagtctcg gctcactgca acctctgtct cccaggttta atcgattctc ctgcctcagc 85020
    ctcccaagta gctgggatta cccatgcccc accatgcccg gctaattttt gtatttttag 85080
    tagaaacggg gcttcaccat gttaggctgg tcttgaaccc ctgacctcaa gtgggcctcc 85140
    cacctcggcc tcccaaagtg ccaggattac aggcatgagc caccgcggcc agccagaaat 85200
    gggttttgga aaaagcacta aacaaaatcg aacttggttt catatgacag ctctgctgct 85260
    aactgtaaca ggggcagacc agttaaccta cttttctgtc ttctgtcagc tgagaattag 85320
    atgattccca aaggcccatt gaactctgaa tgactttaaa tacttcttct taagtgggta 85380
    cacggttttg gtaactgatg ccaggtgatg aatgcatgaa agtgcttaat gaatgaaacc 85440
    ggtaaaatag taggaggaag ctttattggt aaggcagggg tatacctaat agctctctaa 85500
    tttattggta ttgaagtggt taacttttgt ttttttaagg ggggaaaaca ttctaagaat 85560
    aatgaggcaa actgcatatt gcacaagaga ctgttgtctc tattcaacaa ataccttttg 85620
    agtgtccaga gtctgccagg tgctgtgcta ggccctcacg attgagtagt gaaccagaga 85680
    atgtccctgc acccatggag cttattgtct actggggtag acagataata aataagcaaa 85740
    caaatcttct ctcttctccc tttcgctcca tgtaagtgtg tgtgtatagg tgtatactta 85800
    caagttgagt aaagtgttat gaaagattaa gaggagaaat gcattttggt tagatgttag 85860
    aggactcagc aggtgacctt gaaacttaga gctgaaggat cagtaggagg taactagaga 85920
    ggccagggaa tcgcatgttc aaaggccagg aggcaagaaa gagcatggtg cccttcaaga 85980
    gaggaaagaa ggctactgtg actggagcat agatgtaggc aagtgttggg tgattgagag 86040
    ctctacgggc catggttagg ttttattcct aatgccgaga tgccaaacat ggtggttcat 86100
    atctgtaatc ccagtatttt aggaggccga ggcaggaata tagcttgaac ccaggagttc 86160
    aagaccagcc tgagcaacat gagacctgta caaaacattt aaaaaattgc tgggtatgat 86220
    ggtgcacacc tgtggtccca gctactcagg aggctgaggc agaaggatca cttgagccta 86280
    ggaggtggag gctacaatga gccatatttg agtcactaca ctccagcctg gatgacaaag 86340
    tgagaccatg tgtcaaacaa aatacagaaa gaatattaat ttaaaatttt gaaagaggag 86400
    tgatctgaac ttatatctta aaaagatcat tctagggcat ggtggctcat gcctgtaatc 86460
    aagggctttg ggaggctgag acaggaggat cacctgaggc cagttcgaga tcaacctgta 86520
    cagcatagag agactccatc tctacaaaaa gaaaaaataa atagctgggt gttgtgagtt 86580
    attcaggagg ctgaagcaga aagatcactt gagcccagga gtttgaggct gcagtaagct 86640
    atgatcccac cactgcaaca cagtgagatc ttgtctcaaa aaaaaaaaaa aatcattcta 86700
    ggtgcttttt ggaggctgga tgtggtaaga gtagaagctg gagatggtcc tgttagggat 86760
    tcgattcaga ctttaaatac catcaatgca ttgagtccca aatttacatc actacgttgg 86820
    atccttgccc ctgaatccag actggtatat ccaactttag gttcagtttg tatctctacc 86880
    tgaccaatat agaggtgtcc agtcttttgg cttccctagg ccacattgga agaagaattg 86940
    tcttgagcca cacatagagt acactaacgc taacaatagc agatgagcta aaaaaaaatc 87000
    gcaaaactta taatgtttta agaaagttta cgaatttgtg ttgggcacat tcagagccat 87060
    cctgggccgc gggatggaca agcttaatcc agtagatacc ttcaacttac aatatctaaa 87120
    attttatgcc agatttagtc attttaaacc tgctcatcag tttttctcaa gaagtagtat 87180
    tttggctttt tttcttttct tttttttgag atggagtttc gctcttatcg ttcaagctgg 87240
    agtgcagtgg cggatcttgg ctcactgcaa cctccgcctc ctgggttcaa gtgattctcc 87300
    tgcctcagcc tcgcaagtag ctggaattac aggcatgcgc caccatgacc agctaatttt 87360
    tggagacagg gtttcaccat gttggtcagg ctggttttgt actcctgacc tcaggtgatc 87420
    tgcctgcctc ggcctcccaa aggctgggat tacaggcatg agccaccgct cccggctgca 87480
    tttttggatt tttagttgct cagcccaaaa ctttagtaca tctttgaacc tcttctttcc 87540
    tcctactcta tatctgatcc atcagcaaat ctgttaggtc tacctcacac atatcgaaat 87600
    cctaccacgt ctcaccatct gtgacaatta acaccctggt ctaggcagtc atctctgtta 87660
    agattgagtg gttaaggatg tcctctaagg agatgacatt caaatcttag cttaaatgtc 87720
    aagagggagc tggttttata aagattgagg aggcagcatt attttgccat aggcttccat 87780
    ttggtttcca ttccattctt gatacttatg gtatatattc aaaacaaatg cacagaaaca 87840
    gacccaggta tattgggaat ttcggatata gagttcctag ttgggaaaag atagactgat 87900
    ctgtaaatga tgctagttat ccatcatctg gcaaaaaata atttcctgcc tcctctcata 87960
    tatctcagat caacagactt tttctgttaa gggccaaatc ataaatattt taggctttcc 88020
    agaccatatg gtttctgtca cactctcctt tatccttgaa gccatagaca atatgtaaac 88080
    aaatgggcat ggctgtgcta cgataaaact ttacttacaa aaactggtag tgggccagtt 88140
    taggcatggc cagcactttg ggaggctaag gcagatggat cacttggggt caggagtttg 88200
    agaccagcct ggccaacatg gtgaaaccct gtctctacta aaaatacaaa aaatagctgg 88260
    gcatggtggt gggtgtctat aattccagct actctggagg ctaagacaca agaatcactt 88320
    gaacccagga ggcagaggtt gcagtgagct gagatagcac cactgcactc cagccagggt 88380
    gacggagtct taaagcaaaa caaaacaaaa ggtagtgggt tgtatttggc ccatgggctg 88440
    tagtttgcca atccctgatg cagaaacaaa ttccaggtaa ataagagcct ggaatgttaa 88500
    aaaaacaaaa cttgaagtca tgtagaagaa caggtagggg gaacaatcct gatctcagga 88560
    taggaaggga tattgcttaa aataagacac aggaaaatat aatccatgtt gtgtaaattt 88620
    gactacgtta aaacttaaaa ctttcgccaa gcgcggtggc tcacgcctgt aataccagta 88680
    ctttgggagg ccgaggtgag cagatcacca ggtcaggaga ttgagaccat cctggctaac 88740
    acggtgaaac cccgtctcta ctaaaaatac aaaacattag ccgggcgtgg tggcgggcgc 88800
    ctgtagtccc agctacttgg gaggctgagg caggagaatg gcctgaaccc gggaggcgaa 88860
    gcttgcagtg agctgagatc gcgccactgc actccagcct gggcgacaga gtgagattcc 88920
    gtctcaaaaa aacaaaacaa aacaaagcaa aaaacctaaa actttcatac aataaagtat 88980
    acctaagata cttctagaag agaagattta catccaggac gtgtatggaa tttctgcaag 89040
    taataagtaa aagacaaggg acatgaagag gcagttcaca aaagaggaag ccaaaatgac 89100
    caataaacat gaaaggatgt ttaacctcaa aggaaacaag gaaatgaatt aaaaacatca 89160
    aatgccattt caaaactagt aagttggcaa aattaaaaat accaaggatg agaatatgaa 89220
    gcatggctat atgagtgcat ggaatggtac agtcactttc attaaaaatg cacataattt 89280
    gttttttatt tatttttttg agacagtcta tgtcgcccag gctagaatgc agtggcatga 89340
    tctcggctca ccacaatctc tgcctcctgg gttcaagcaa ttctcctgcc tcagcctcct 89400
    gagtagctgg gattacaggc acatgccaca acgcccggtt aagttttgta tttttagtag 89460
    agacagggtt ttgccatgtt ggccaggctg gtctcgaact cctgacctca ggtgagctgc 89520
    ttcccaaagt gctgggatta gaggcgtgag ccaatgctcc tggctgaaaa aaatgcacat 89580
    aatttgttac ctagcaattc catgtctaga ggcttatcct agagaaattc ttgcttatat 89640
    gcataggaag acgtgtacta gaatgttcac tagttgaatg tttaagtgaa aattaggaaa 89700
    taaagtaaat gttcattaac aggaaaatga gtaaaggtat atttataaaa caattaagta 89760
    gctaaaatga ataaactaga gctgcgtgaa tgaactagaa ctggttcaat agtcatgtca 89820
    gattattgaa tgaatacagg tcagatatgt atagagtgtc atttgtgtaa ttaatttttt 89880
    tttttttttt gagatggagt ctcactctgt tgcccaggct ggagtgcagt ggcgtgatct 89940
    cagctcactg caacctccac ctcctgggtt aaagtgattc tcctgcctca gcctcccgag 90000
    tagttgggat tacaggcatg caccaccatg cccagctcat tttcctattt ttagtggcca 90060
    cagggtttca ccatgttggc caggctggtc ttgaactcct gacctcaagt gttccaccca 90120
    acttggcctc ccaaagtgct aggattacag gcgtgagcca ccgtgctcag ccatttgcgt 90180
    gatttttaaa gatgtgcaga ataatgccat taaaaaaaat acacatacat gtatatatat 90240
    acacgtttgg ctgggtgtgg tggctcacac ctgtaatccc agcactttgg gaggctgagg 90300
    caggaggatc acttgagccc aggtgtacaa gactagcctg ggcgagatag caagacccca 90360
    tctcaacaac agaaaggata attaggtatg gtggcatgag aggatcactt gagcccagga 90420
    gttcgagtgt tatcaggcca ctgcactcta gcctggacaa caaagcaaga ccgtgtctca 90480
    aaaaaataaa aataaaaagt atttgtatgt ggtcatagtc aaaaaacgta catggaagga 90540
    aaatgtcttt atttatttat ttattttttt ttttttaaga cagagtcttg ctctgtcacc 90600
    caggctgggg tacagtggtg taatctcagc tcaccgcaat ctcggcctcc cgggttcaag 90660
    cgattcttct gcctcagcct tctaagtagc tgggactaca ggtacccgcc accacaccct 90720
    gctaattctt gtgttttcag tagagacagg gtttcaccat gttggcaagg ctggtctcga 90780
    actcctgacc ttaagtgagc cacccgcctt ggcctcccaa agtcctggga ttacaggtgt 90840
    gagccactgc gcttggccag gaaatatcta atttagtaag tatttatatc tgggaaagga 90900
    agggtcaggt ggtgattcat aggaactcta aagtctatgt ataatactta gggggacaga 90960
    aggaaataaa gcaaaatgct gatatttgat tgttgagttg tgtatatgtt agaagtataa 91020
    cataggagat ctgattgata gtaggagaat gtttttaggt ggtaaaagtg gaaccgtggt 91080
    ggtttgtttt ggcagtagaa tcagttggtc atagtttgta tgtggaaggt aataaacaga 91140
    ccatgttaag gatgacttcc ggaattttgg tctgagtagt gggtggatga cagtgtcatt 91200
    catgagggaa gatgaagact gaggtaggaa caggtttggg agaagatgac atgttccctt 91260
    ttagacaagt ggaattatgg aagatggcag gtaggtggtt agctatatga atttgagata 91320
    aaagatttag gatggagata taaatttagg agtaacagcg tatctatggt attgtaagcc 91380
    ttaagaatgg gtaggatcag ccaggaaata cagatgtata tgcagaagag aggagtcaag 91440
    gaagccaaga caagttaatg tttaaagtga gtgatgtagt ccatgggcag atgctgctga 91500
    gagggctgca aacaccagtg accctacaac atttttaaat gtcgtcttcc tgacagcagt 91560
    gatcagtacc tgcaacgatc ttatttattt ttttcatgtt agtctccaca cacttgaatg 91620
    tagacttttt gaaggcaaaa tcattgcctt ttctgagctg ggagcatgtc tggcacatac 91680
    caagcactca acagttgatg tattgacttc atccagatac tctgagggcg agttatttcc 91740
    tgctactagc ctttcacctt tcaatgttta agagcacaaa tacagagatg ggcacgtttt 91800
    ggcatttctt attttgataa ccttttcctg gtaagatttt ttaatgttga aaaaaaaaaa 91860
    caagaaaaga gggttaaaaa tagtcttatg tcagatcctg tgatagaatt cacacttggc 91920
    ttaagctgct gggcaccttc ctatcttgga tgtcatatta gcttatctac agcagaattt 91980
    ttactgtttt atgtagtaag gaagcaatta tatgattatt ttacagacaa attattcttt 92040
    atcttttatt tttttagacg gagtctctct ttgtctccca ggctggagta cagtgtcgcg 92100
    atctcggctc actgcaacct ccgcctcctg ggttcaagca attctctgcc tcagcctccc 92160
    aagtagctgg gcttacaggt gtccgccacc acacccagct cattgttttg tatttttagt 92220
    agagatgggg tttcaccatg ttggccaggc tggtcttgag ctactgacct caggtgatcc 92280
    acccgccttg gcatcccaaa gtgctggaat tacaggcgtg agccaccgtg cctggcccag 92340
    acaaattatt atactctgag tgttagaggc ttaggatgtt ttcacttgat gctatgggag 92400
    gaataagtaa taagatatga tacacaacca aagacctttc ttcactatgc ttctagtagc 92460
    tagtactatg gatgacacat ggtaataata ttggttagca tttgtcctca atttactgtg 92520
    ctagttactc ttctaagccc cttacaggta tatatttttt ttcatcaata atcctctaag 92580
    gtagttttta ttattgacct aattttataa atcaagaaaa ttaagaccca gagaagtaag 92640
    taacttgtcc aagatcacat ggcttataag tggtagagcc agaatttgac cccagatgtt 92700
    gtgactacat tgtctctcca taagcaggtt caactctttt gactggatgc tgttccaagg 92760
    tcacttcctt agagaagcct ttgctgacaa ctaccctcct gtgccctcct ccaaggctgt 92820
    ccattgttct agaactttga atactcatct tagaataaag ctggtctaat ttttacagtg 92880
    ttatagaatg gatctctgac tgcaaaagtt ggtcataatt atctttttat gttctagtga 92940
    aaggcaaaga acaagagaag acctcagatg tgaagtccat taaaggtaag ttctgccctt 93000
    ggcagtccac tgcattaaaa agtgatgtgc tttgcatttg tgagttcttt aatcctgtta 93060
    tactctctct tttggcatta atcatttctg ccttatttta taattactta tgattttgat 93120
    ttatttccct ctttaacctg tataatgctt taacatctag catataataa gtaggctttt 93180
    tttttttttt tttttttgga gacggagtct tgctctgtta cccaggctgg agtgcagtgg 93240
    cgcgatcttg gctcactgca agctctgtct cccgggttca caccattctc ctgcctcagc 93300
    ctccccagca gctgggacta caggtgcacg gcgccacgcc tggctaattt tttgtatttt 93360
    ttagtagaga cagagtttca ccatgttagc cagtatggtc tcgatctcct gaccttgtga 93420
    tccgcccgcc tcggcctccc aaagtgctgg gattacaagc gtgagccacc gcacccggcc 93480
    gtaagtaggc tttttttacc ttaattttat ttttttgaga tggagtcttg ctcttatccc 93540
    caggctggag tgcagtggtg ccatctcggc tcactgcagc atccacctcc cgggttcaag 93600
    cgattctcct gcctcagcct cccgagtagc tgggattaca ggtggccgcc accatgccca 93660
    gctaattttt gtatttttag tagagacagg gtttcaccgt gttggccagg ccagtctcaa 93720
    actcctgacc tcaagtgatc cactcgcctt ggcctcccaa agtcctggga ttacaggcgt 93780
    gagccaccat gcctggccat aagtaggctt ttactgagcc ttgtgtgtat tggctatcct 93840
    agtgattaca gtgaaccagt gcccttctta ttaatcacac atttaattgt tccctaaaag 93900
    tgattagttc actttattta tttagtaaga caaaaaatga agaatactct taactgagca 93960
    gtctgttaac tgtaggaaag cactgacact tataaggctt agttttctgt catttatcca 94020
    gaagtatggt tgattacagt ttttactttt ttatttgaat gaacaacctt aatttaaaat 94080
    atattttgtt tattttttgt tgggatcgat acattgtcct tgtttataga ttagagcatg 94140
    ctttttaaag atgctgtatt actcactgat tttatttgtc cagtgtacag agattgaagt 94200
    gggaaaatta taatggaaat tgtttccata gtcattacat attaatttca tcaatttatt 94260
    tccataaaat ctgtagattg ctacttattt agatttttcc ttcaaatgtt tttatgttgt 94320
    attgcttgca ctgagtattt attctatatg ctcaatttgc tggagaagaa gactaattat 94380
    aacttaggca agttgtaaaa ttagggaaaa aagtaaggta ccttacagcc tagtttactt 94440
    atttcttatg taaagccagt tagattccac attagttcaa actgccttct ttgagcaaaa 94500
    cttgattggc agtgataaag gcttaaagcc cttctcaagc agagacctgt aaagactaga 94560
    tctgactgta gtagaaggaa ggaacttaga tgtttcaggc agtgagaaca ccagtcttcc 94620
    actctaaact ttgccactaa cagtatgacc ttgggaagtt gtaactttct tcagattctt 94680
    catttgttga atggggggat tggcctagct aatttctaaa tctctactgg gctaaaaaat 94740
    tctgtgctta tactctgatt atgaagtaca taatctgtgc ttaacattca ctgacttatc 94800
    cttaggataa tacagaagca gtacaagaaa cagcccctca agatgtttgc agtctggtta 94860
    gaaagacaaa cttatacaca gaacagtagc aaatagacca aaataataat agctgccatt 94920
    tatagaacac ttcttctgtt ctgggcatta gacaaaaact gactataacg gtgaacaaaa 94980
    aagacttagg tcctgccctc attgaactta cagattagta ggggagagga acattaatca 95040
    agtaattcca cagatggctt agcctagatt ggtagtgatg gaagtaaaga gatgtgaacg 95100
    gacttgaaaa aaaattcgga ggcaaaatgg atagaagttt attattgatt aaatatgagg 95160
    tgtgagagag agggatattt aagattgata cctaccttct ggcttgccta acagaaccaa 95220
    aacaggaaat tatatgttca gttttgttat gttgggtggg aggtgctttt gagtcattca 95280
    tttatatatg ttatatatgt tattttatat gcatagtaat tttaaggtct gagttttaaa 95340
    ccaaaggtta gagagtgatt ttttagagtc tagcaaacct aagttgaaat cctgcctgtt 95400
    gaaatggctg tttactagct cattaaccta gggcaaagta ttcaacttgt tttcattttt 95460
    gtcttcatct ctaaaatgag gaaaatatgg tcttacaaga ttgtcctgag agatagatga 95520
    aataatatcc aaaaaaaaaa aaggtacata gagaaactcg tatagtgcct ggtatatagt 95580
    aggtcctcca ttggtagcta tcattatcta gttttaacat agccttcagt ttgttgaatt 95640
    agtcaaactg agtgaagcac tgcaaggaat tcagaggaat ttgagatcaa caaatgattt 95700
    ctgaagttta gggaagactt catggcaatg acacttacct tgtataaaag ttgaagaata 95760
    agaaagattt gaatgagaga ttctttctct tctccctacc agcccagctt cttatttgag 95820
    gatatattgg gcaaaggggc cttcagacaa gtagagggag atttttacag aaagattgag 95880
    atgaaggtat agaaggctgt aaagaccaga aaagagaatt gagacagagg aagcaggaag 95940
    ccactgtagg tttttgagca agatattgat gctgtaagta tggtgtttat gaaaggttag 96000
    tctggaagag atttgcagga tggagacccc ggaagttttt ttgttataat acagaaagac 96060
    ttgcactgag ggtgaggtgt taaaaataaa caggtaagta aatgtttaaa catcttgaag 96120
    gaaaagtcaa caaatcttgg caagtaaaca gataacagtg aaaaagaatg ggaccaagat 96180
    tttgagtttt ggagactggt ggattgaaca gacagggaaa ttgagaggag aatcagatga 96240
    tgatgtttta agttgatatt tagacagatt gtgcttgaga tggtaaagtc aatgtgggtg 96300
    ggaatgctta gtagcgagta atcagtgata caagaccaaa gcccaggtca aagacaagtc 96360
    acagatacag atcagggctt tttcatctgc tccacagagg tgtaccctag gagctgttgc 96420
    aaacagtcca tgtggagggt gtgagtaaga tgtttccctt gaatttgcca gaattacttt 96480
    tttgttgttg ttgttgtttt ttctgagaca gattctcgct ctgttgccca ggctggaggg 96540
    cagtggcgag atcgcgcagc tcactgcaac ctctgcctct cgggttcgag tgattctcct 96600
    gcctcagcct cccaagtagc tgggattaca ggcttgtgcc accaagccca gctaatttct 96660
    tttgtatttt tagtagagat ggggtttcac catgttggcc agactggtct cgaactcctg 96720
    gcctcgtgat ctgcctgcct cagcctccaa aagttctggg attacaggcg tgaaccactg 96780
    cacccggtcc cttgttaagt ttattttggt gggaagcaaa ggaggtttca gcttttaaaa 96840
    agtttgaaaa ttattgctct ggtaataatt aaagatttga gagtaaatat gctttctagc 96900
    agaaagaata aaagaagaac agatagcctc aagaagggga gccaaagaag caggctatat 96960
    ctgacacact gggtgttgat aaatgggtat taaaagaatg agagcaatga gcagatagaa 97020
    gaggaaatta ggagagtata ataccatgga gaccaagaaa gatagactat caggaaggag 97080
    tggtaaaaat aagttactag ttctaagaga gatgttaaga gggaccgggg aaagccttgt 97140
    acaaatgagt tagtagcatt ttacattata tacatctaat taagaaacaa tgcgagagtc 97200
    tcaccattcc tatagactct tacttgtact tgtctgaaca cgaaaactgg cttttgttta 97260
    taaataagct aaaaattatt ttgctccaat ttctcatgaa aataaaaata aaccttcttt 97320
    taacattgaa aaaatagttt gaagacagtc actcttcatt ttgtaattcc cacaactatt 97380
    attgaatgac tgaaattatc tttattctga agccaaaggg gtgatactga tatttcttca 97440
    gactactaaa aatatatttt atgaattttt agtgtgcttt atcttttttt gttttttttt 97500
    ttgagatgga gtttcactcc cgttgctcag gctggagggc agtggtgcaa tctcagctca 97560
    ctgcaacctt cgcctcccag attcaagcaa ttctcctgcc tcggtctccc aagtagctgg 97620
    gattacaggc acctgccccc acacccagct aattttttgt atttttagta gagacagggt 97680
    ttcaccatgt tggtcaggct ggtcttgaac tcctgacctc aggtgatcca cccaccttgg 97740
    cctcccaaag tactgcgatt gcaggcatga gccaccatgc ctggcctgag gaatattttt 97800
    ctaggttccc cccaccccaa gcatttattc tgcaatttta gttttgttcc taaagcaagc 97860
    aaggtttaag gatttaaaaa taatccgtat tttagaatgc tttctggctt tgttactttt 97920
    tatccacagt agaagttctc agagaatgat ctccctcttt taatttaact ttttggcaca 97980
    gtattttgag aattataaat aatattagaa tgttttctgg ctgggtgtgg tggctcatgc 98040
    ctgtaatcct ggctacttgg gaggctgagg caggagaatc acttgaacat gggaggcaga 98100
    ggttgcagtg agccgaggtc atgccactgc actccagcct gggtgacaga gcaagactct 98160
    gtctgggaaa aaaaaaaaaa aaaaaaagag tgttttcttt cctattttcc accacttgat 98220
    taagttactt ttcctcttaa gtattttttg ctgagtatgc tgacttaaga gtaatgttac 98280
    aaaatttaat ttttaaagtt ctctgaaagc ccctttatga gagttttagg ctatcaaatt 98340
    gtgtttaatt cttaacaatt ttttgaaaaa ttatagcttc aatatccgta cattccccac 98400
    aaaaaagcac taaaaatcat gccttgctgg aggctgcagg accaagtcat gttgcaatca 98460
    atgccatttc tgccaacatg gactcctttt caagtagcag gacagccaca cttaagaagc 98520
    agccaagcca catggaggcc gctcattttg gtgacctggg taagtaacta tcatttttta 98580
    ttaacttgta ttagaaggat ttgagtacaa tatgtgaaac ttctgtcata ggatacagaa 98640
    ctatataatt ggaaagtgct ttggaaaaaa tgtatttaaa ataacagcta caagtataat 98700
    gggtagctgt gttgtgttcc tgtaaatata gaatataaag catgcccagt agaaaaacaa 98760
    gcatttccag aagaaatata tctgatcact aaatataaat atatgaaaaa gatgtctcac 98820
    tttattactg agggaagtgc aaattaaaat aatcagttaa tgttctccta acacattagc 98880
    atatttttta aagtttgaca atttgaatgt cagtgaagat gcagggaaat acccctccta 98940
    tttagtgata atataatctg gtgaagactc tttggaaagc aatttggaaa tcagtataaa 99000
    atatgcatgt catttaggcc actctttcta agacctagcc ctcagatatg ctcattcata 99060
    tgtgcaggtg tgtatgtgtg tgtgtgtgtg tgtgtgtgtg tgtatatgta tgtatgtatg 99120
    tatgtatgta tgtatgttga aggctattca ttatagtatt gtttgtgata gcaaaaaatt 99180
    atggacaaca tataaatatc tgttataggg aaataaccaa attgtggtat acgcatgctc 99240
    tggagtataa tatagccatt tgtttctatt tatttatttt cttgagacag ggttttactc 99300
    tgttgcccag gctggagtgc agtggtatga tcatggttca ctgcagcctt cacctcctgg 99360
    gcacaagcca ttctctcgcc tcagcctcca gagttactag gactgcaggc atgtgtcacc 99420
    acacccagat aattttttaa ttttttgtag agacagggtc tcactatgtt gcctaagctg 99480
    gtctcaaact cctggcctca agcaattctc ccacacaggc ctcccaaagt gctgggatta 99540
    ccaacgtgaa ccaccacacc tggttcagtg tagccattta gaaatctaaa aaagacgtgg 99600
    gaaaatgtct aaggcatgtt taaatgtgag aaaagcaagt cacagtatgc atggtaaaat 99660
    ccgttatatt aaaataagtt cttccaaaac aaaaacatat gcaggagacc tttattttgt 99720
    cagtatttct tacccaaatt tctgcactta gaaaattgca tgtcatgttg tcataagttg 99780
    aaaaaaagat ccatgaacca atggacttct aataaaatca gtcctgcttt tgacatctct 99840
    ctctactttt gtgtatattc aaaccagagt gtcaatgtgt ttgtggggca cacttagcaa 99900
    taatacatag cagacaaaat gcatatagct cagagagtaa aattgtaagt tttgctagat 99960
    cactcataaa ttgctgatga gaatttaaaa tggtgcagat gctctggaaa acaggcagtt 100020
    tctttctttc tttttttttt tctttttgag acagggtctc actctgttgc gcaggctgga 100080
    gtacagtggc gtgattacaa ctcactgcag cctcaccctc ctcaggttca ggtgatcctc 100140
    cctcagtctc ctgagtagct gggactatag gcatgcacca ccacgcctgg ctaatttttg 100200
    tatttttttt tttttttttt gtagagacgg ggtttcgcca tgtttcccag gctggtctca 100260
    aactcctgga atcaagcgat ccacttgcgt aggcctccca aagtgctggg attacgggcg 100320
    tgagctactg tgcctggcct aggcagtttg tttgtttgtt tgtttgtttg tttatttatt 100380
    tgtagacgga gtctcacagg ctggagtgca gtggcccaat ttttggctca ctgcaacctc 100440
    cgcctcccag gttcaagcta ttctcctgcc tcagcctcct gagtagctgg gatgacaggt 100500
    gcctgccata atgcctggct gatttttgta tatttagtag atatggggtt tcaccatgtt 100560
    ggtcaggctg gttttgaact cctgacctca ggtgatcagc ccgcctcggc ctcccaaagt 100620
    gctgggatta caggcatgag ccgtcatccc tggctggtgg tttcttatga cgtgaaacat 100680
    gcaattacca tatgacctag cagttgcact ctgtatttat cccagataaa tgaaaactta 100740
    ccttccaata aaaacctgtg cacaaatgtt catagcagct taatattgaa aaactggatg 100800
    ttcttcagca ggtgaatgaa ctggttcatt cataccatgg aataccattc agcaataaaa 100860
    aggaacaaac tgttgataca tttaaccacc tggatgaata tcaagggaat tatgctgtca 100920
    gacaaaaacc agtccctaaa gactacatat agtatgattc cgtttggata atattcttga 100980
    aatagagaaa ttaagagaaa tgaaaagatt agtgtttgcc agatgttaga gacagggagg 101040
    tgagaggggt aagtgggtgt agttataaaa gtgcaacatg agggatcttt gtgatgttga 101100
    agttgtatct tggcagtgga tgcagaaatc tcaatgtgat aaaattacaa agaactaaaa 101160
    acaagaatga gtatagataa aactggggaa atctgaacaa gttagagtgt tgtatcactg 101220
    tcagtatctt agagtgatat tgtactatag ctttgcaaga tgttaccatg ggagaaacta 101280
    aagtgtacaa gggatctcta ggtattatta tttttttaga gatggggttt cactatgttc 101340
    cccaggccgg tcttgaactc ctgggctcta gtgatccgcc tgccccagcc tcctaaagta 101400
    ctggaattac aggcgtgagc gaccatgcct ggccctttca gtattgtatc ttagaacttc 101460
    atgtgaatct agcattatct catagaattt aattaaaaga aattgtaaac ctcacagaag 101520
    atcagaattt cctcaagttt gtgatgttga caaagatgaa ctagttgaca ctgacagtaa 101580
    gactgaggat gaagacacga cgtgcttcaa aaaaatgatt tgaatatcaa tggattaaga 101640
    agaactcttt tgacaaattg atgaaaccct cagtcagttt tataagaatg cccatcttta 101700
    tgatcatgct atgaaagcca atttttaaaa aaattttttg tctttcctaa caattagctt 101760
    gtggttataa tttaaattta gttaaatata agataaatga ttttttatta agtttagttt 101820
    catttttcaa ggtacgatct caaagctact ctttaaccta ctatgaatga ataatgctga 101880
    gttcataaca tctttgtaga tatatccaca attttccctc aggataagtg cctacaagtg 101940
    gaattactgg actgaaaata atgcagtttg ctaagacttt gctatctgtt cctgaatgct 102000
    cctccaaaaa ggttttgcca gtttacatcc tcatgaccag cgaatgagag tgttgcctat 102060
    tttcctgtgc ccttgttact gcttaataat ttttgaaaaa aatctaattt gacagacaaa 102120
    aatgcatttt atgttaattt gcttttctgg gatttttaat gaggttgagt atagttttta 102180
    atatttttat tggccccttt ggaactagta tcataagttt tttttcttaa gaatttatgt 102240
    agtctgggct gggcgcagtg gctcacgcct gcaatcccag cactttggga ggccgaggtg 102300
    ggtggattgc cgaaggtcag gagtttgaga ccatcctgac caacatggtg aaaccgaatc 102360
    tctactaaaa gtacaaaaac tagctcagcg tggtggcggg tgcctgtaat cccagctact 102420
    taggaggctg agtcaagaga atcgcttgaa cccgggaggt ggaggttggt tgcattgagc 102480
    cgagatcgcg ccattgctct ccagcctagg caacaagagt gaaaagtctc aaaaaaaaaa 102540
    aaaaaaaaaa aaaaaagaat ttacatggtc tgaattgcca ttaaaagaga tatgagaatt 102600
    attgagtaac aaataacttt ttaataattt aggcaagttt tggacgattg tactttgttt 102660
    agaaaccaaa agcatagtat ttgtagtttt tttatttact ttagttgcta ggaagtaaac 102720
    tttattcaag gtctctggta ccagttgttg ctaaaagtga ttgactaatc tgtcaatctg 102780
    aaattatttg ttgctgaact gctaattctt ttgcttctat cttttaggca gatcttgtct 102840
    ggactaccag actcaagaga ccaaatcaag cctttctaag acccttgaac aagtcttgca 102900
    cgacactatt gtcctccctt acttcattca attcatggaa cttcggcgaa tggagcattt 102960
    ggtgaaattt tggttagagg ctgaaagttt tcattcaaca acttggtcgc gaataagagc 103020
    acacagtcta aacacagtga agcagagctc actggctgag cctgtctctc catctaaaaa 103080
    gcatgaaact acagcgtctt ttttaactga ttctcttgat aagagattgg aggattctgg 103140
    ctcagcacag ttgtttatga ctcattcaga aggaattgac ctgaataata gaactaacag 103200
    cactcagaat cacttgctgc tttcccagga atgtgacagt gcccattctc tccgtcttga 103260
    aatggccaga gcaggaactc accaagtttc catggaaacc caagaatctt cctctacact 103320
    tacagtagcc agtagaaata gtcccgcttc tccactaaaa gaattgtcag gaaaactaat 103380
    gaaaagtgag tatgtgattt tcttgtgtgt acatatgtgt ctcactttct ttttttaatt 103440
    tactaagcag aacttcagat gaggaataaa atgattggaa tatttttttt ctcctctaac 103500
    tacttgtaaa tttgggagaa tttggagagt gtagtagagt cagatcagtg tatggaaaag 103560
    gagcaggagt gactggacct tctaagaagt gtgttatcag aattagtaaa tgaagggtca 103620
    aatgtcctac ttttcccctc cactgatttt gacatcaaac cattatccac atagccttat 103680
    ttcctccctc ggtcttaatt ttattaatat tttactgcac tttgcagata aaatttttaa 103740
    aaaattttta aaaattgcca ataagtgaca tttattaagt tcagtgctta gtgtatattt 103800
    ggattttatt tattagtcac aagacctttg tgcaggtagt aggcatgatt atcttttttt 103860
    ttttgagatg gagtcttgct ctgtcgccca ggctggagtg caatggcgcg gtctcggctc 103920
    actgcaacct ccgggttcat gccattctcc tgcctcagcc tcccaaatag ctgggactac 103980
    aggcgcctgc caccacaccc ggctaatttt tttgtatttt tagtagagac ggggtttcac 104040
    catgttcgcc aggatggtct cgatctcctg actttgtgat ccgcctgcct cggcctccca 104100
    aagtgctggg attacaggca tgagccaccg cgcccggact gattatctta tttacacatg 104160
    agaaaaccag ggcttagaaa ggttaggtaa cttcctctag gttgtacagt aaatgtggac 104220
    ctagaagcat tttgacaaga gcacctgttt ttttttcttc tctattagtt tagaaattat 104280
    atactcttaa ttatcacctg ggattttgat tagacagcct tcatgttctt tttcatctta 104340
    aatgttcttt gtgtcttaaa gggctaagtg atttcttcag atcttttagt tcactcattc 104400
    tcagtgaact aaaatgaggt ctaatctgct actgaatcaa gttttcagca tgttatttcc 104460
    ttcctccctc cctccctcct tccttccctc aaccaggctc ccgaggagct gggattacag 104520
    gcgcccgcca ccactcctgg ctaattttta tattttagta gagacggggt ttcaccatgt 104580
    tggtcaggct gatcttgaac tcctgacctc aagtgaccca cctgcctcgg cctcccaaag 104640
    tgctgggatt acaggcatga atcaccacac ctgacggcat gttattttca tcgcaaagtt 104700
    actgtaagct gggagaagtg gcacacactt gtactcccag ctactcagga agcttaaggt 104760
    gagaagattg cttgagccca ggagttttga gaccaacctg ggcaacacag caagacccca 104820
    gctcaaacaa agaaaaaaag ttattgaatt ttttatttct atggatcatt ttttgtagtt 104880
    tcttattcct ttcacccttc attcccactt ttgatcccat cttttattta tttagtttta 104940
    ttaaatgtat atttgtctga taattctgct atctacagtt ttttgtggac ctgactcagc 105000
    atttctttgt ttcttcggat tcagactgtt ggtggcttgt gattttagtg atttttggcc 105060
    gtgaacatgt ttcttggact tttgtctgtg ggaattctct gtgtactctg tataaattaa 105120
    gttacttcag gtgttttgca ttttcttttg ccatgcacct ggggcctggg tcactaccct 105180
    tctggtacca cttaaaactg aatttttgtc ttgggtgctc gtactgatcc tgtatgagta 105240
    caggtttata cttactgtag aaatatggtg tttgattatg gggtattgtc ccagatggtg 105300
    ctggagtatt aatatgctct ctgttaaact taatgtgttg tccctgtaaa actccaaaat 105360
    tctgaattcc agaatactac tggccccaaa tgtttaagat aagggcactg cctgtatttg 105420
    tttctgcctc ccactatttt ccttagttta acacaaactc acctttttaa aaaacatttt 105480
    gagagaattc agtattggga agagtttcta acctgtttct ggaaatggaa gtccaaagtc 105540
    tgtttctgta attgtttttt ttttgagatg gagtctcact ctgtcaccca ggctggagtg 105600
    caatgacgta ctctcagctc actgcaacct ccacctcccg ggttcaagcg attctcttgc 105660
    ctcagccccc tgagtagctg ggattacagg tgcccaccac catgcctggc tgatttttgt 105720
    atttttagaa gagatggggt ttcgccatgt tggccaggct ggtcttgaac tcctgacttt 105780
    gtgatctgcc cacctcagcc tcccaaagtg ctaggattat gtttctgtaa ttgtaataca 105840
    tttattgttt ttagaaactg tctttgcttt agtggtaatt ttcaataaaa atagaaatag 105900
    cagtggagtt attaaaagag cattagttac atttttccct ttttcattat cttcaaatat 105960
    tatatatagt aagtttgacc tttttaaaat gtatacttgt atcagtttta acacatacat 106020
    agattcctgt aactgtcacc actataaggg taaagaacag ttagttcctt cacctttgaa 106080
    gtcaagcccc acctctatcc caacacttgg caaccgctga tctttctccg tctcaatagc 106140
    tttgcctttt ctcttttttt ttcttatttt tttttttgag acagcgtctt gctctgtcgc 106200
    ccgagctgga gtgcagtgag gcaatctcgg ctcactgcaa cctccgcctc ctgggttcaa 106260
    gcagttctcc tgccttagcc tccctagtag ctgggattat aggcacgcac caccacaccc 106320
    ggctgatttt tttgtatttt tagtagaaat ggggtttcac catgttggcc aggctggtct 106380
    caaactcttg acctcaagtg atccacctgc ctcggcctcc caaagtgctg ggattacagg 106440
    cgtgagccac tgtgcccaat caggactttt tttttttaaa tttacattca acttgtcatt 106500
    tttttcttgt atggattgtg ccttcagagt cacacctaag agccctttgc ctaagcaaag 106560
    gtcatgaaga ttttctcata tgtttccttt taaaagtatt gtggttggcc aggtgccatg 106620
    gcttatgcct gtaatctcag cactttgaga agctgaggtg ggcagattac gaggtcagga 106680
    gatcgagacc atcctggcta atgcggtgaa accccatctc tactaaaaat acaaaaaaaa 106740
    aaaaaaatta gccgggcgtg gtggcgggca cctgtagtcc cagctacttg agaggttgag 106800
    gcaggagaat agtgtgaacc cgggaggtgg agcttgcagt gagccgagat cgcgccactg 106860
    cactccagcc tgggcaacac agtgagactc catctcaaaa aaaaaaaaaa agtattatgg 106920
    ttttacactt tacgtttaga tatatatctt ttttgagtta atgtcgtata agtatgaggg 106980
    ttacgtcaga ttttttgttt tttgtttatt tttacatatg gatgtctagt tgttctaata 107040
    ccatttgttg aaaagacaac ctttactcca ttgaattgcc tttgtacttt tgccatattt 107100
    gtctaggcct gtttttggac tcctttttct gtttcatgat gtgtgtgtct attcctttgt 107160
    taataccaca tggtcttaat tactgtatag taagtcttaa aattgggtaa tgctggcctt 107220
    ataaaacgaa ttgggaagtt tttattttta ctcttatttc cattttctag aagagattgt 107280
    gtagaattgg tgtcatttct tctttagata tttggttgaa ttgggaagtg atgccatctg 107340
    ggcctagggt tttgtttttt gtgtgtgaga cagagtctca cttctgtcac ccaggttgga 107400
    gtgcagtggt gagatcttgg cttactgcaa cctctgcctc ccaggttcaa gttatcctcc 107460
    tgcctcagcc tcccaaatag ctgggattac aagcgtgtgc caccatgccc gactaatttt 107520
    tgtattttta atgcagacag ggtttcacca tgttagccaa gctggtctcg aacttgtgac 107580
    ctcaagtgat tagcccacct tggcctccca aagtgttagg attatagatg tgagccaccg 107640
    tgcctggcag gggcctaggg ttttcttttt cagagtattt taaactatga attcagatta 107700
    tttaatagat ataggactat ttaagttatc tgtttcttct tgagtgaatt tttactgtag 107760
    tttatggcct ttgagtaatt aattgtattg aattgtcaaa tttatgagcg tgtaattatt 107820
    tatagcattt cgggtttgta gtggtatccc tcttttattc ctggtgttgg caattgtgtc 107880
    ttgtttttct ttgtcagatt gtatagggat ttattagtct tttcaaagaa ctagcttttg 107940
    ttttgatttt tctgttgttt tgttttcaat tttattgatt ttctgctctt tattatttct 108000
    tttctattat ttctgcttgc tttgggttta ttttactctt ttttttttct ccaagttgct 108060
    taaagtagaa acttagattt ctggtttgag acctttcttt tctaagataa gcatttaata 108120
    ctgtaaattt ccttctaacc actgctttag ttacaccccc acaaattctg gtattttgaa 108180
    ctgagcacaa atgaaatgtt ctaatttccc ttgaatctta ttcttttacc aatgaattat 108240
    ttagaaatat gttatttagt ttgcaagcaa ttggagactt ttttcctgtt atttttctac 108300
    catttatttc tcatttcatt atattatggt cagagaatat attttgaatg atttcattta 108360
    ttaattttta aaaataacat taaaaaattt tttaaaatgt gaatatacca catacagtat 108420
    aaagattgta cattctgttt ttggacagtt ttctataaat gtcaagttga tttagttggt 108480
    taatgatggt gttcagtttt tctttattct tgctgatact ttgtatgcag ttatatcact 108540
    ttattactca gaagagtgtt gaactttcca actacaattt ttttttccaa ttttactttc 108600
    agctctatct ggttttgctt catgtatttt gaggctctgt tgttaggtgt gtacacattc 108660
    aggatgatat cttctgggtg aattgcctgt tttatcatta tgtaattccc tctttatggt 108720
    aattttcctt gttctaagat cagaaatatc tgttgtccaa tttatataga cactgcagct 108780
    ttcatttgat tagtgcttgc atggcatatc tttttccatt tttttacttt tgatctacct 108840
    ttataattct atttaaaggg ggcttcttgt aggcagcata tagttgggta gtgttattta 108900
    tttatttatt tatttattta tttatttatt tattgagaca gagttttgct cttgttgccc 108960
    aagctggagt gcagtggtgc aatcctggct taccacaacc tccacctcct gggttgcagt 109020
    gattctcctg cctcagcctc ccaagtagct gggattacag gcacgcgcac catgcctggc 109080
    tgattttttg tatttttagt agaaacggat tttcaccatg ttagccaggc tcgtcttgaa 109140
    ctcctgacct caggtgatcc acctgctttg gcctcccaaa gtgctgggat tacaggcgtg 109200
    agccactgca cccggctgag tcatgttatt tttaatcttt tctcacaata cagggttttt 109260
    gttggtaaat ttaattattt taatataaat tttagtataa ttatttacat taaatgtaac 109320
    tgttgcactg gggtatttat aatgtgtaaa tataattatt ggtattaata taattatatt 109380
    actcataata atattaatat ctttggattt agattaccag tttagtatat gtttttctgt 109440
    ttctccctct ttgatttccc cttttttgct tttttttttt ttttaattct tatttttttt 109500
    tagtatttgt tgatcattct tgggtgtttc ttggagaggg ggatttggca gggtcatagg 109560
    acaatagttg agggaaggtc agcagataaa catgtgaaca aggtctctgg ttttcctaga 109620
    cagaggaccc tgcggccttc tgcagtgttt gtgtccctgg gtacttgaga ttagggagtg 109680
    gtgatgactc ttaacgagca tgctgccttc aagcatctgt ttaacaaagc acatcttgca 109740
    ccacccttaa tccatttaac cctgagtggt aatagcacat gtttcagaga gcagggggtt 109800
    gggggtaagg ttatagatta acagcatccc aaggcagaag aatttttctt agtacagaac 109860
    aaaatggagt ctcccatgtc tacttctttc tacacagaca cagtaacaat ctgatctctc 109920
    tttcttttcc ccacatttcc cccttttcta ttcgacaaaa ctgccatcgt catcatggcc 109980
    cgttctcaat gagctgttgg gtacacctcc cagacggggt ggcagctggg cagaggggct 110040
    cctcacttcc cagatggggc agccgggcag aggcgccccc cacctcccag acggggcagt 110100
    ggccgggcgg aggcgccccc cacctccctc ccggatgggg cggctggccg ggcgggggct 110160
    gaccccccac ctccctcccg gacggggcgg ctggccgggc gggggctgac cccccacctc 110220
    cctcccagat ggggcggctg gccgggcggg ggctgccccc cacctccctc ccggacgggg 110280
    cggctgccgg gctgaggggc tcctcacttc gcagaccggg cggctgccgg gcggaggggc 110340
    tcctcacttc tcagacgggg cggccgggca gagacgctcc tcacctccca gatggggtgg 110400
    cggtcgggca gagacactcc tcagttccca gacggggtcg cggccgggca gaggcgctcc 110460
    tcccatccca gacggggcgg cggggcagag gtggtcccca catctcagac gatgggctgc 110520
    cgggcagaga cactcctcac ttcctagacg ggatggcagc cgggaagagg tgctcctcac 110580
    ttcccagacg gggcggccgg tcagaggggc tcctcacatc ccagacgatg ggcggctagg 110640
    cagagacgct cctcacttcc cggacggggt ggcggccggg cagaggctgc aatctcggca 110700
    ctttgggagg ccaaggcagg cggctgggaa gtggaggttg tagggagctg agatcacgcc 110760
    actgcactcc agcctgggca acattgagca ttgagtgagc gagactccgt ctgcaatcct 110820
    ggcacctcgg gaggccgagg caggcagatc actcgcggtc aggagctgga gaccagcccg 110880
    gccaacacag cgaaaccccg tctccaccaa aaaatgcaaa aaccagtcag gtgtggcggc 110940
    gtgcgcctgc aatcccaggc actctgcagg ctgaggcagg agaatcaggc agggaggttg 111000
    cagtgagccg agatggcggc agtacagtcc agcctcggct ttcacaactt tggtggcatc 111060
    agagggagac cggggagagg gagagggaga cgagggagag cccctttttt gctttctttt 111120
    ggattatttg aatttttcct taaatttatt tatcttactt atttatttat ttttttgagt 111180
    gattctcctg ccacagctcc caagtagctg ggactgcagg catgtgccac tacacccagc 111240
    taattttttt gtatttttag tagagacagg gtttcaccat attggccagg ctggtcttga 111300
    actcttgacc tcaagtgatc cacctgcctc ggcctcccaa agtgctggga ttacaggcgt 111360
    gagccaccat gccctgcctt tttctagaat ttatatattg agttcttgat tgtatctttt 111420
    tatgtaggct ttttagtggc ttctctagga attacaatat acatactttt cacagtgtac 111480
    tcacatttaa tattttgtaa cttcaagtgg aatgtagaaa acttaaccac cataaaaata 111540
    gaactaggga tgaggttaaa aaagagagag aaaagaaatg taataaagat ttaataacac 111600
    cgtttttttt tttttttctc tttttttttt gagacagagt ctctctttct gttaccaggc 111660
    tggagtgcag tggcgtgatc ttggctcact gcaacctccg cctcctgggt tcaagtgttt 111720
    ctcctgcctc agcctactga gtagctggga ttacaggtgc gcgccaccat gcccagctaa 111780
    tttttgtatt tttagtagag acggtttcac tgtgttggcc aggatggtct cgatttcttg 111840
    accttgtgat tcgctctcct cagcctccca aagtgctggg attacaggcg tgagccaccg 111900
    cgcccggcta agtctttaaa tatttttttg acattgcact ttttctcttt tccttctagg 111960
    attttagtaa cccaaatgtt agttttgtta ttgtttggca ggttcctgag gctttcctta 112020
    cttctttaaa tttttttttc ctgttgttca gcttcgaaaa tttctattca tctgtcttca 112080
    aattcactgg ttctttcccg ttatttccat tctgttattg agtctttgta gtgaatttta 112140
    aattttgttt attatgtttt ttagttctaa aattttcttt ttttgtgtat gtcttatact 112200
    ttgctcctga aactcttatt tgtttcagga gtgatcttat ttcttagagc atggttttag 112260
    tagctactta aaatttgttt tatcatccca gcatatgtgt cctcttgatt gtcttttctc 112320
    ttgtgagata atgggatttt ctggttcttt atatgacaat taattttgga ttgtatcttg 112380
    gacagtttga cttacgttac atgattctga atcttgttta aatcctgtgg aaaatattga 112440
    agtttttgct ttaacaagca gttgacctag ttaggttcag tccacaaatt ctaagcagca 112500
    ttctgtcggc tctggttcca tcatcagttc agttttgtat cttatctgct tatgtgcctt 112560
    tctgtgtcca gtctgggacc tggccaatgg tcaggtccca aagcctttgt acacttttag 112620
    aagcagggcc atgcacaccc agctcacgag tggccccggg agtgcacata caactcgacg 112680
    ttttcatggg ctccttcttt tctgtgatgt ccctgacacg ttctgccttc taagaacctc 112740
    cctttatccc tttcctgttg tctggctaga aagtcagggc tttagattcc ctatacttca 112800
    gcacacttcc tgtagctatg tcaacctctg tggccacgac ttcttcttct tgggactgca 112860
    gtttctcttg tcagaaagta ggattcttgg agctgctgtc attgctgctg tggctgctct 112920
    gatgctgcct gggagtcgaa ggagagaaag gaacaaaaca aaacaaccca ggggatttcc 112980
    tccactctct ttgatccgtg agagccccct ttcctgttcc tcagaccaga aatagagggc 113040
    ctgtcttgga acttcttctt tgtgcatctg gtgtgcagtt tcagcttttg agtccaggcc 113100
    aggaggtgct ggacaaactt gtcaggagta cggaggtact gcaagttctg attacttttc 113160
    tcagtccacc tgcttccaag tccttggatg catttgtcca ttgttttgag ttgcattcca 113220
    tgggagagac agaagagtgt gcttatttca tcttgacata cttattagga tttcatatca 113280
    aatcaacgga tgatattctc tatattaatt tgctgttttc cctttagcaa gcacattagg 113340
    aaaataacac tttaacaccc gcctttggtg gtttctgtca taattattaa tacttgactt 113400
    tttttttttt tttgagacgg agtctcactc tgtcctttga ggcattgtcc ccataaactt 113460
    ttggtaaagc atcaataatt ttatctttca tccacacaag cttcaccata aatttgatgt 113520
    ttattcttcc attttagcag aattcatgtt gctccaatag gggctgtctt caaactgatg 113580
    ttttctcctt cttagtgcct cagagtagat cctgttcaga tacgttataa caggttaata 113640
    tgagtttatt ttggtgtaaa agtactttga aattcatgca tagttttttc atcatatgca 113700
    ttttccatag ctttgaacac ccccatgtaa ctctcctctt ccacaaacca aacaatgaaa 113760
    aagcaccttt gtgatggaag tttattttgc aataggaact cacagtgatc taagccctgc 113820
    tattcatgaa tataattcat tactggagtc caagttgctt tttggttttt gaagttctct 113880
    tcttcccttg caggtataga acaagatgca gtgaatactt ttaccaaata tatatctcca 113940
    gatgctgcta aaccaatacc aattacagaa gcaatgagaa atgacatcat aggtaagcag 114000
    tgcttgaaac tatggcaaaa aaaaaatgac aaaaaatgca cagaactgac aattttcgtt 114060
    attgactaag ataatttttt cttaacatgg aatttagcag ttcccttcct aatttgtttt 114120
    ctgagtattt tttatatcgg attatagctc actttaaaag tttctcggct gcattcggtg 114180
    cgagggtctt tgcctgggcc agatgggctg cagtgtagcg ggtgctcagg cctgcccgct 114240
    gctgagcagc cgggccggcg ggcggctacg ctaaccggca cagaccaccg gatggactgg 114300
    ccggcagccc cgcaccagtg cacgaagtgg gcgggacaga aacttctggg gttggaagtc 114360
    cagtgaggct aaaagccggt accaaagtct ctaggcatca gggctgcagc ccaagagtct 114420
    cacgaccagt gggcaactgg atggccagac aggtgtctca gtggtggcct ctccgtctca 114480
    gggcttcatc ccacttctca gtgggcctga cgtccctggg caccctggat gtctacctgc 114540
    attagccaga gccatcacat ggcctgtgac ttgccttttt ttgccagttg attgtgccac 114600
    acacagtgtc atttctgtgt catttggcac agctggaggt gcaaggagga gggcagcctc 114660
    atgtccagtc ccagtttcac gtaactttat tcttctgaat aaagacaatt tgctaacctt 114720
    aaaaaaaaaa aaaaaaaaaa agtttttctt atatgttgga cccaaattct taggctttaa 114780
    cctgaataac aatgacagca agatcaataa atagtacaca tttattaaac actcactgtg 114840
    tcccagacaa tattccaagc actttttatg gatagactca ttttaacttc taaagaactt 114900
    tgtgggataa atacagttat tttatagatg aagaaactga agcacagaga agttaagtgc 114960
    tttgtccagg gtaacagctc agatatggca gagtcaggat ttgaaactag accctcacat 115020
    accttaactg ctgtgctgtg gcagtgtttt tcatactgta ggttgggacc agccttctct 115080
    tatgccctca ccccctgcca aaaaaaaaaa aaaaaaaaaa aaatatatat atatatatat 115140
    atatatatat atatatatat aatatatata tatataaaat atatatatat ataaaatata 115200
    tgtattagta tatatgcata tatagtatat attatatatt agtatatata ctaatatata 115260
    atatacatat tagtgtgtgt atatatatat atactagaat aaaaaaatca aagtatctca 115320
    gagtagtaag gacaaacatt tcagaaaaat gttttcatta tatatacatg tatgtatgtg 115380
    tatgctgatt caacaaatat atttcttata ggttatagca aaatagtttg aaagctttta 115440
    ctgtgtttta tcaggaagac cttaggtgaa cgtatattca cagataaaag aggttattta 115500
    ttcattcaat aaatattaca ttctcataag tcctaatatt atgtattttt attcttcaaa 115560
    aaagttagta tttgtgattt atgaaataag acatgttctt gcacttttag cagatctgtc 115620
    ccgatgttgg gcttctttaa tccttagtgt gggtgctttg cactcactca ctgctgggga 115680
    cagcaagacc cctgttagtc tcagctgtgt ttcttaaatt ggcccactgt accttccagt 115740
    tagctattct ggggtccatg tcatgttggc tccattttcc ttttctttct cccacacaga 115800
    tacctataac ggctataaca taggcctggt ggctgttggt ggcttatccc tatctgcttg 115860
    tatttaaggg gtactgtttc actgagtttt gctgacagat gttgtcatga gatttgaggt 115920
    tttctgtgtt gttgctctat ttttatgtgg gaatttgcta ctatcatcat ccctagacca 115980
    gcttttccta gtaatacaac agggatgttc tgactgatta gagtttgcct gtttgaagaa 116040
    ttggttggct agtgattttt ttttgagggg agtctgtacc agttaatagc ctgactggcg 116100
    tgtggataaa aaggaagcag tttcaagtca aataaaacac ttaaaatgaa accacactgc 116160
    aactctcttt cttttactta agcttaatca aattaatgat gatgtaatcc catgaaggaa 116220
    aagtcttctg aaggatcaag ttgataacat tttgtgatca aagaatttga gaaaacctct 116280
    atcccagtgt ctatcattat atattttagg atgttaatta cctgtgtggc tttaggcaag 116340
    tcatttttcc tccttgagcc ccattcttaa tcctgtccaa attatttgtc tcctcttgca 116400
    gttggactat tttaatatag ctgtccttca agtgagtttt gttcaaagga gccttcactt 116460
    tagctcttac tgtgtaccca ctttgcatag tcttgtttta aatgtaatcc ttggattttt 116520
    ggtgttgcta actaattact gtttttatgt gaggatttag agtgatccag aatctatact 116580
    tgcactacct ccttcatctt ccacaaatgt ttgaagtggt agaattttta aaaactttga 116640
    aggtacagct gacagaattt gctgatggtt tggaagtgag tggtatgaga gggaaaaaaa 116700
    ggaataaagc atgactgcat tttttgtttg tttgtttgtt tgtttttgag acggagtctc 116760
    actctcgcca ggctggagtg cagtggcgtg atcttggctc acggcaacct ccgcctcctg 116820
    ggttcaagcg attcccctgc ctcagcctcc caagtagctg ggactacagg cgctcgccac 116880
    cacgcctggc taattttttt ttttgtattt tagtagaaac ggggtttcac cgtgttggcc 116940
    aggatggtct ccatctcctg acctcatgat ctactcacct tggcctccca aagtgctgag 117000
    gttacaggca tatatataag catataaagt gtgttatagc atacaaacag gtatatatat 117060
    aaacatgcag tccacacagc tgataggaat gaggcagtag tgaaggagaa gttgatgtag 117120
    gagaggggac agttgttaca ggaaagaagt ctggaggcag aagggatgaa ttccagtgct 117180
    cacatagaag attgcttaga tgggagcaag gacaatttat ctagagtcac aggaaagaat 117240
    gcagtacacg ggtagagatg caggtgagtt gaaagatgtg agagatgatg gaaataattt 117300
    tctgattgct tctatattct caaggaagca ggaagcaaag tcctcagcaa agagaataga 117360
    agaggtgtta aatatttgag aaaggagatg tactgtagaa aaaaaaaaaa ctcagtttct 117420
    ccttctgaac tctcacaaaa cagaaccctt ccatgactct agttgtgtgg ggttttttcc 117480
    ctgtcagcta ccaattctgc agatgattgt tcagtgaaca ccaactgggt gtcctctaag 117540
    tcagttcagt tctcacactg tttacctgga gatagcatca gatcccacag attgaggact 117600
    ctgtcccaca agactgcctc cacttcagat gccagtctca agtacaagtt gtggcctgtg 117660
    cttctgactg accttctata aattggagtt cccacagtcc cctccttggg ttcaataaat 117720
    ttgctagagc agctctcaga actcagggaa atgctttaca tatatttacc catttattat 117780
    aaaggatatt acaaaggata cagattgaac aggcagatgg aagagatgca tgggcaaggt 117840
    atgggagagg ggcacagagc ttccatgcac tctccaggtc atgccaccct ccaagaacct 117900
    ctacagattt agctattcag aagcccccct ccccattctg tccttttggg ttttttgtgg 117960
    agacttcatt atataggcat gattgatcat tggctattgg tgatcagctc aaccttcagc 118020
    cccctcatcc cgggaggttg gtgggtaggg ctgaaagtcc caaacgtgta attctgcctt 118080
    ggtctttctg gtgattagcc ctcatcctaa agctctttag aggccacagc cacaagtcat 118140
    ctcattagcc ttcaaaagaa tccagagatt ccatgaattt taggcgctgt atgctaagaa 118200
    actggctaaa ggccagttgc aatgtctcag gcctgtaatc ccagcacttt gggaggctga 118260
    ggcaggagga tcgtttcagg ccatgagatc aaaaccagcc tggtcaacat agtgagaccc 118320
    ccttacaaaa aatttaaaaa ttggccaggc gtaatagctc ttgtctgtag tctcagctac 118380
    tcagaaggct gaggatcact gagccctgga gttgaaggca gcagtgagcc atgatcgtgc 118440
    cactgactcc ggcttgggtg acaaagtgag accttgtctc agaagaaaaa ggaaaaaaaa 118500
    aaaactgggc aaagactaaa taacatattt cacagtatca cagatttgta ttgtctagga 118560
    aagtgaatgt aaacagacca ggacactagt atgatccctt ggtttcatga aggtcccact 118620
    aaagtcatga acacaaagtg agactaggca tcatgttata tggtttttcc agccatgttt 118680
    aacagctagc taaatagcta attgtttcgc tgcagtttat tttagcagtt ccttatttta 118740
    gcacatttca tgttttaaaa tttctaccaa taacatttta ataaactttt ttacagataa 118800
    cttcacaaat ccataatttt ttaagttaca atcccagaaa tagaattgct cattgaaagg 118860
    gtatgttcat ttttaaagtt atgctagaaa ctgccaaatt gccttcagaa aaaggtgttt 118920
    gtatccccac taacactagt gttagttttc ttgtgccctt gctcaagtat acatattatt 118980
    aaaaacaatg ttgggccagt ttactagata aaaggtgtag tgcctcctta ttctaatcta 119040
    tttgattact agtgagtatg tatgtctttt cacgttggtc attttatgtt tgttcctttg 119100
    tggattgtca tgtcctttgc tcatttttct tttggaacat ttcttagtag tttataagag 119160
    ctcttggtat tttaatgata gtaacctttt aactgtcatg catgctgcaa atcttttttc 119220
    tgtttgtttg cctttgtatt ttgtttttgg agggtttcta tgtataggaa ttaaatttta 119280
    tgttgttaaa tcttttgatt tctgcttttg catatgtact tcaaaagact ttctatttta 119340
    agatcaagtg ttacctgtat tttcttttag ttctatttaa aacctcttaa tttatatgcc 119400
    tgtgctgtta actcccaagt tgattcacaa gtgtgtatac atagtttgaa tttagtggca 119460
    atttaattat ttacaacttc ttttgcagca aggatttgtg gagaagatgg acaggtggat 119520
    cccaactgtt tcgttttggc acagtccata gtctttagtg caatggagca agagtaagtt 119580
    agttcatatt ttcacattgt gcatcctagg gaatttgggt tcattgttag gaatgggctt 119640
    cactcagcta aaaacaaagt atttttgaga atttaaatat tttggatatt tacaagatca 119700
    tataaagcat actctatctt ggttaacagt ttcttttaaa tataaattat gtgaactctt 119760
    aaaattttca ttttcatttt caatgttaat atttcctaag ttaaaataat ttgtttttag 119820
    ttctgaaata atttggggag tgattgagtc tgtagtgatt atgactatta gaattggttt 119880
    atttatttaa ataatgcatg tcttcagatg gctctcctaa tttgttagtt aggctttaag 119940
    ctaaatggat gctatataac taaatccaca tagatttgtt gaaatggctc cagaggtttt 120000
    ttagatttat tactgctatg tgcccttaaa aaaaatctat tcattctttc acttaacatt 120060
    tatcagaaga gtgctctgtg taagacgtgg ttaggcatag tgccagtctt gaaggaagtt 120120
    acagcctaat aaaagacata gggcatgttg tttggttact gtaatatgaa gtggcatgtg 120180
    ttaaatgtca ggggagaact acaaagtcat aaaaaggtgg gagagattac atacaggtaa 120240
    aggaatcagg aatgacacca tggggagtaa ggtagtgttg acctaggcct ttaagataca 120300
    atagggacag tatggaaaga gtatattttt cccacttaaa ctctttcctt ggtcgttccc 120360
    tcaaattttc ccttttgtcc atgtgcaggc actttagtga gtttctgcga agtcaccatt 120420
    tctgtaaata ccagattgaa gtgctgacca gtggaactgt ttacctggct gacattctct 120480
    tctgtgagtc agccctcttt tatttctctg aggtaaagtc tgcatttctt ttcacactct 120540
    attcgagcat tccagcctct aactatcaat gctggggccc tgtctatagg aaataacaca 120600
    gaagagccaa gtcatttcca aaaagatgta tcattgtttc aagttgtttc tgatggcaag 120660
    agtaatttaa taatatatta gagagaacat gaaaattcaa tgtattaaat aactctaatt 120720
    ttgagaaacc taattaaact actgcatgta agagagtgca tgtttttaat tatttggagc 120780
    tattttaaaa ccacagaatt tgaaacttgc ttccagtgca taaattgcag accagacttc 120840
    agaagagaaa aaaagtagta aattttttct tatgctcatc atttttactt tagtcacttg 120900
    ataggattgc ccagtgaaga agcatttgca acagacaatg agtatattaa tctttttgag 120960
    gcatacagtt tagtataatg ctctttgtta ggcttcaaca agtgaaatta ttttgttgga 121020
    aagcaaatga ctattaagta gaaagaggat tcccagtctc acaaagcagt aatttagaca 121080
    ctcgattctg cctctttaca agaatacagg tactcagttg atttgttttc tcactccctt 121140
    tctttgctat aagtttaaat caacaatttg tttaggttaa tatgtcctca tggaatggtg 121200
    gaaatgatca gatataaaat atttggtttg gttagtttac tctttatatg tttgctggca 121260
    aggaaccaca aatccagttt agtataattt ttactctagt tcactaaaag tttgcatcca 121320
    gctgtgtagg tagtgtttgt ttcttgttaa cttttttttc gtctaaaaga atactttaaa 121380
    acttttcaat ctcaaatgac tgtaacttgc tgacaggtgt taacagaaga agtagatctt 121440
    tttgtttttt gcttatgacc tgtattttaa tatttgagct tatagattag agattgtgag 121500
    agaaatctgt ttatagtctt attttccctt gtgtattttt tcttcctagt acatggaaaa 121560
    agaggatgca gtgaatatct tacaattctg gttggcagca gataacttcc agtctcagct 121620
    tgctgccaaa aagggccaat atgatggaca ggaggcacag aatgatgcca tgattttata 121680
    tgacaagtga gttatattga tagatggatt cagcagatac ttattgaaca tttgatatgt 121740
    tttgtggaaa taaagatgaa taaactcagt ctctgttgtc aaggagctca caggaggcag 121800
    cataaaagct gcttttatat ggtgtttgta aagctttggg ggttcttaga acaaaagttt 121860
    ctgctgggaa aggggaggtg tatgtggggt aaacaggatg gcaatggtgg tgttcaagga 121920
    gtgtttccca gaagagagat tttgtttgga tcccaaagaa agaagggaat tttgctaccc 121980
    agagaaggca gaaaacaaca ttctaggcaa aggcattggc ccagaagcca tggaaacgta 122040
    ggggaaagtg gcactttcaa gaaacttgag tttagataat caaaggagtg gggaataaat 122100
    atgaggatgc tggtactaat tggaatagat tgtaagggac cttgaatgcc tatttatggg 122160
    tatattatac tttctgtata aatctgctca ggcacgttgt taattagttt tttattagtt 122220
    ttcactgaaa atgagaggat ggaaacatca tacagtaaac aaaattgaaa atatctggtc 122280
    aggcagatga tgagcttgtg gccagctctg taacgtatgg tattcttttc atttaacttt 122340
    tcttactctg taaaaaaagt aattcgtggt cgggcacggt ggctcactcc tgtaatcaca 122400
    acactttgag aggcagaggc aggtgaatcg cttgagccca ggaatttgag accagcctgg 122460
    gcaacatggc aaaacccgcc tttactaaaa atacaaaaat tagctgagcg tgatggcgtg 122520
    cgcctgttgt cctagctact taggggcctg aggcagaagg atcacctgag ccttgggagg 122580
    tcgaggctgc agtgagctgt gatccactgt actccaccct gggcagggca gtagagtgag 122640
    accctgtctc caaaaaaaaa aaaaacaaca aaggtaattt gttatttgta tccttaagca 122700
    aatgctaaag gggtaacttg gggatagaga aaagtccaca gatgttaggg tttgaagaca 122760
    ctaatagtat ctaggccagt ggttcctgaa cattagtctg tgggctcttg ctgggctgtc 122820
    tgcataggaa tcacctgaga gcttattaaa aataggtttt caggctggtt gcggtggctc 122880
    acgcctataa tcccagcact ttgggaggct gaggcaggcg gattacttga ggtcaggcgt 122940
    tcaagaccag cctggccaac atggtaaaac cccgtctcta ctaaaaatac aagaattagc 123000
    caggcatgat ggcacacacc tgtaatccca gctactcagg aggctgagga aggagaattg 123060
    ctcgagcccg ggaggtggag gttgcagtga gcggagatca tgccactgca ctccaggctg 123120
    gctgacagag ggagactctg tctcagaaaa aaaaaaaaaa ataggttttc agtctgggta 123180
    ccggtggctc acacctgtaa tcccagcact ttgggaggcc aaggcaggca gatcacttga 123240
    ggtcaggagt ttgagaactg cctggccaac atagtgaaac cttgtctcta ctagaaacta 123300
    caaaaaatta actgggcatt ttgacgggtg cctataatcc cagctactag ggaggctgag 123360
    gcaggagaat tgcttgaacc cgggaggcag aggactgcat ctcaaaaaaa aaaaaaaaaa 123420
    aaaggtttcc agtccccctg tctcagaaat tctgattctg caggtttgag gtgtgaccag 123480
    gaatctttat ttttagaaga cataccagat aattctgata aatagccagt ttagggatgt 123540
    agtctaattt tcctattttg caagtaagga aaataaggcc cagagaggta atgattttct 123600
    caaagtcaca gaacaagtta gtggcagaat ttggactgga atgcagttct taatgttctg 123660
    tccagtgttt attctggtac agtatgtttg tagaaggtat tacgtaagaa acattgttat 123720
    atagatgttg agataggaag agtttacatt tagaaatttg gtctaaaatg cctgaacatt 123780
    caagtcgtgg aggagtattg accaacttac tcaatacaac ataggagatt cacattttgt 123840
    tacaaaaatg ctgatttaaa aggagagttt tctttttttt cttctttttt attttttgag 123900
    atggagtctt gctctgtcac ccaggctaga gtgcagtgac acgatctcag ctcactgcaa 123960
    cctccacctc ctgggttcaa gcggttctcc tgcctcagcc tcctgagtag ctgggattac 124020
    aggtgggggc caccacgccc agctaatttt tgtattttta gtagagacag ggtttcacca 124080
    tgttggccag gccggtcttg aactcctgac ctcaagtgat ccacccacca ctgcctccca 124140
    aagtgctggg attataggcg tgagccactg tgcccagcct gcttgttttt gtatcatata 124200
    tatgcatcat cataatcatg cattatcaac ctttgtattt ctgtcaggac atagaaacca 124260
    ttagagtgct tggaagagag cctttttttt tttctcgcat ttaatgcttt ttttggtatt 124320
    catttcataa tcagcttacc aaaacattac ctgcattata ccccatcaag gtagaaatct 124380
    ttgtgttatc aatattggtt actccctttc cacaccgagt catcagtaag tcctgttcta 124440
    tccaaatagg tcatatgcat ctagctcacc cctcagtgct gttttgtttt gaatttgtac 124500
    atgtttactc ctgatgcctt gtagttatga tgatgtgttc ttattttatt ctgtgcatac 124560
    aagttctcag ctcgcttttt agggaaaatg accatgtctt cctttcctat aaattccttt 124620
    ctatctatca agtcctcaac agagaatagg tacccataaa tatgtgattg ttagtttctt 124680
    tgcctcagtt gtagtctgat ccttacagct tttaaacaac agtagagttc accgtcaaga 124740
    actaaggatg gttggcaggc agatagaaag gtagcaagtt gacccaacta tctctgggga 124800
    agtgggaaca aagaaaggtt acatcagcac tgtcatcaca tagctctata gttctaggcc 124860
    tgcaggctca atcaagtagc cttgtataag attctctgga ggaggtgctg aaagttgctt 124920
    atacttgcta tggaatttga ttttacttcg gatatctttt taccataggt acttctccct 124980
    ccaagccaca catcctcttg gatttgatga tgttgtacga ttagaaattg aatccaatat 125040
    ctgcagggaa ggtgggccac tccccaactg tttcacaact ccattacgtc aggcctggac 125100
    aaccatggag aaggtaaccc agaacttcaa acgtatcaaa ctacaagaag ttttattggt 125160
    agaactcata aaatataagg tgggaaaacc aagcagaata gcacagtgga aattgaagca 125220
    gtccagcaaa gtgattaaga gcagaggcct tgagtctggc ctggtatgta cagtcacgtg 125280
    ccacataaca ttttagtcaa cagtggactg cgtgtacgat ggtcctgtac gattataatg 125340
    gatcaaagct ggtagtgcaa taataacaaa agttagaaaa aataaatttt aataagtaaa 125400
    aaagaaaaaa gaaaaactaa aaagataaaa gaataaccaa gaacaaaaca aaaaaaatta 125460
    taatggagct gaaaaatctc tgttgcctca tatttactgt actatacttt taatcattat 125520
    tttagagtgc tccttctact tactaagaaa acagttaact gtaaaacagc ttcagacagg 125580
    tccttcagga ggtttccaga aggaggcatt gttatcaaag gagatgacgg ctccatgcgt 125640
    gttactgccc ctgaagacct tccagtggga caagatgtgg aggtgaaaga aagtgttatt 125700
    gatgatcctg accctgtgta ggcttaggct aatgtgggtg tttgtcttag tttttaacaa 125760
    acaaatttaa aaagaaaaaa aaaattaaaa atagaaaaaa gcttataaaa taaggatata 125820
    atgaaaatat ttttgtacag ctgtatatgt ttgtgtttta agctgttatg acaacagagt 125880
    caaaaagcta aaaaaagtaa aacagttaaa aagttacagt aagctaattt attattaaag 125940
    aaaaaaattt taaataaatt tagtgtagcc taagtgtaca gtgtaagtct acagtagtgt 126000
    acaataatgt gctaggcctt cacattcact taccactcac tcgctgactc acccagagca 126060
    acttccagtc ttgcaagctc cattcatggt aagtgcccta tacagatgta ccatttttta 126120
    tcttttatac tgtattttta ctgtgccttt tctgtatttg tgtttaaata cacaaattct 126180
    taccattgca atagtggcct acgatattca ttatagtaac atgtgataca ggtttgtagc 126240
    ccaaaagcaa taggttgtac catatagcca aggggtgtag taggccatac catctaggtt 126300
    tgtataagta cactctgtga tgttagcaca atggcaagca gcctaacgga aattctgttt 126360
    attgattgat tgattgattg attgattgag acagagtttc actccattgt ccaggctgga 126420
    gtgcagttgc acagtcttgg cacactgcaa cttctgcctc ccaggttcaa ccaattatcc 126480
    tgcctcatcc tcccaagtag ctgggattac aggcaggcac caccatacct ggctaatttt 126540
    tgtattttag tagagacagg gtttcaccat tttggccagg ctgttctcga actcctgacc 126600
    ttaagtgatc tgcctgcttt ggcctccgaa agtgctggga ttacaggcat gagctaccat 126660
    gcctgggcag taactgaaat tctctaatgc cattttcctt atctgtaaag tgacgataat 126720
    atgcacgttt acctcaaagt tactttgatg attaaagtaa ggtaatgtat ataaaataca 126780
    tattaacata gtacctgaca catggtaagc atcaaaaaat gttaactact tttattacta 126840
    ttattattac gtatttttaa ataattagag agcagtatca aaaattagct gggcgtagtg 126900
    gcatgcacct atagttccag ctactcagga ggctgaagct ggaggattgc atgagcctgg 126960
    gaattaaagg ctgcagtgag ccgtgttcat gcccctgcac tccagccttg gtgacagagc 127020
    aagaccctgt cttgaacaat taaagaaggc attatgccgc aacgttagct tagaaatgat 127080
    ccacatatat caccagtaac tgtcaacagg attggaaccc tagttttggg tattatgatc 127140
    acaaggtatt attaatagct tattaataat aaagcgttgg ctaggcacgg cgactcacat 127200
    ctgtaatccc agcactttgg gaggccgagg tgggtggatc acctgaggtc aggagtttga 127260
    gaccagcctg accaacatgg agaaacccca tctctactaa aaatacaaaa ttagccgggc 127320
    gtggtggtgc atgcctgtaa tcccagctac ttaggaggct gaggcaggaa aatctcttga 127380
    acccgggagg cagaggttgc agtgagctga gatcgcacca ttgcactcca gcctgggcaa 127440
    caagagcaaa actccgtctc aaaaatataa ttataataaa taaataaaag taaagtattg 127500
    atgtttgtga atgatttatt cttctaatga actagaggag atttttccag gaatttcaga 127560
    gccagtgagg ttatgttgct tgtatgtgtc atgtgtatcc aggtgaaaaa acttaattaa 127620
    acgctattat ataataccat acataaaaac tgaattttag gaatactgaa gaatgacata 127680
    tagaagtcaa atcattaaat agctagtagt aaacagaata gagtgtcagc tgttacccaa 127740
    tgatgataat attttcacga ttaaaattaa accttttctg attttaaagg aaaagttcag 127800
    atctgtatca tataaagaat gtaaattttc agggtaataa aattaaaatg cagagagaaa 127860
    aatgcaaaaa tagttcttac tagatgtgtg tatgtaagga acttagacta attttaagaa 127920
    cactgtcaag accctggtag ttaggtagga aaaaagacat gaatgattca ttcaacaaaa 127980
    actttgagta tttctgtgct agatggtagt gttacagtgg taaacaaaat aaatgtgttt 128040
    ctgctatcct ggagcttagt ctacaaaaaa ggtacatatt ggccgggcac ggtggctcac 128100
    gcctgtaatc ctagcacttt ggaagatcga ggcgggtgga tcacctgagg tcaggagttc 128160
    aagaccagct tggccaacat ggcgaaaccc cgtctctact aaaaatacaa aaattaactg 128220
    ggtgtggtgg cggacacctg taatcccagc tactcgggag gctgaggcag gagaatcact 128280
    tgaacctggg agacagaggt tccagtgagt cgagatcatg ccactgcatt ccagcccggg 128340
    ggacaaaagc gaaaatacgt ctcaaaaaaa caaaaacaaa caacaaaggc acgtattaaa 128400
    tacgaacata aatatttaca aattatactg aataagttct catgtttatt atttgcttgt 128460
    ccagttacaa acttttcctt cgtagaatta gaaatataaa taataaacat gagaactcat 128520
    tcagtataat taataattat taaatgtaaa taaaaacatc tatgtacaat taggcattta 128580
    tttaagaatt atttgaaaaa aaaacaatgt ggaaacagat attttgatat attgctagtg 128640
    attgaaattg ataatgttct tttgaagagt aaagtgacca tatatattaa agttaaaatt 128700
    taactcagca atcacacgcc tggtgagtta tcttaaggaa atcagtttga aagtaaaatc 128760
    aatatatgca caaagacttt aacatttatc ataaaccaga aaaatcgagt ttcaaattat 128820
    atcctatgga ctattttctg ctaaaaagta ttaatatcaa ctttatgtaa tactttcgtg 128880
    acaaatattt tgggggagaa aacccaacaa aattacatgc attgtaattt tttttttttt 128940
    ttttttttta gacagtcttg ctccagcgtc caggctggag tgcagtggtg caatctcggc 129000
    tcactgcaac ctccatctcc caggttcaag caattctcct gcctcaggcc tcccgagtag 129060
    ctgggattac aggcgctcac caccatgcct agctaatttt tatagttttt agtagagatg 129120
    gggtttcatc atgttggcca ggctggtctt gaactcctgg tctcaagtga tccgtctgcc 129180
    tcggcctcct agagtgctga gattacaggt gtaagccact gcacccagcc ttatgcatta 129240
    taattttaat ttgtaaactg tacaaaggga taatacttgt agtacaacaa gaagtaaaaa 129300
    catttgttat aggtagttaa catttgtaac cagtagaatt ataggtaaaa tttatttatt 129360
    taaaacagtt ttagttggat ttgatttcaa ctttaaaata atgcttttca tctctatcag 129420
    gtctttttgc ctggcttttt gtccagcaat ctttattata aatatttgaa tgatctcatc 129480
    cattcggttc gaggagatga atttctgggc gggaacgtgt cgctgactgc tcctggctct 129540
    gttggccctc ctgatgagtc tcacccaggg agttctgaca gctctgcgtc tcaggtattg 129600
    actgattgcg tctgccatta gggagaaaag catacacatc ctttccttca catcccagta 129660
    acagatccta ttatttgtaa attttaagtt gtggaaaaaa aagataaaag ccaggcacag 129720
    tggcctgtgc ctgtaatccc agcactttgg gaggctgcgg tgggcggatc acacgaggtc 129780
    aggaattcga gaccagcctg gccgacatgg tgaaacccca tctctactaa aaatacaaaa 129840
    attagccggg catggtggca ggcacctgta atcctagcta cttgggaggc tgaggcagga 129900
    gaatcgcttg aacccaggag gcagaggttg caatgaacca aaatcacgcc actgcactcc 129960
    agcctgggtg acaaagtgag actgtgtctc aaaaaaaaaa aaaaaagaga gaaataaaat 130020
    tagcctactt actatcttct aatcaaagca tttgtggtaa cttaaaatat actgtattgt 130080
    aaagtatcat gctgtttcat ttaggccatt attctatttg aatctgtggc tgtttctctt 130140
    aataaatcaa gtaatatgga atatattcat agcctctgaa gagctcttta tgtaagtatt 130200
    tatttaggat actttttgta aaataagtga atgaattctt aggtctcctt tttttttctt 130260
    ttcttgagac agggtctcct cgctgcaacc tggaaattct gggctcaaat aatccaccca 130320
    ccacagcctc ctgaatagct gggactagag gcatgcacca ccacgcctgg ctaatttgaa 130380
    attttttttt ggccaggcat gatggttcac gcctgtaatc ccagcacttt gggagaccga 130440
    ggcaggcaga tcacgaggtc gggagatgga gaccagcctg gccaacgtgg tgaaaccccg 130500
    tctctactaa aaatacaaaa attagctggt tatggtggct catgcctgta atcccagcta 130560
    cttgggaggc tgaggcagga gaatggcttc aaccagggag tcggaggttg cagtgagccg 130620
    agatcacgcc actgcactcc tgcatggtga cagagtgaga ctccatctca aaaaaaattt 130680
    tttttttaaa tgatggagtc ttgctgtgtt gctcaggctg gtcttgaacc cctgacctca 130740
    aatgccgcct gcttcagcct aagtttcttt tttttttgta aagagacagg gtcttgctat 130800
    gttggccagg gtagtctcaa actcctggct tcaagcagtc ctcccacctt ggcctctcaa 130860
    agtgctggga ttacaggcgt gaaccactac ctataatgtt gtgtttcact caaggccttt 130920
    tgatttcgtt ttgcattacc gtgccacatt gtgcatttcc ttgacctttt ttgggttttt 130980
    tggagtgctt tcatatgtta aaccatacct gattctcctc aaaatcacac aaagtagaat 131040
    atcctaagac aagaaatcta aggaggcata aagaagttaa ctggttttat taaactcaca 131100
    cagtaaatga tagagccaga aatattcccc ttctagtgtt cttcaccatc agcttaatgt 131160
    agcataataa ttttctaatt actgttgaca aataaataac cctttgaatt ttcaatactg 131220
    ggccttggat aaattttcct aatttgtaag agagtattat cgtattgcca tttacaaagc 131280
    tctcctgagt atctttttct tctgttaagt ttacctagga gataaactgc tgagtatggt 131340
    tgccattttg gttttttgat ataggttaga atgtcttggt tttttttttt tttttttttg 131400
    gtttttgttg ttgtcattgt ttgagacagc atcttgctct gtcgcccagg ctggagtgca 131460
    atggcacgat cgtggctcac tgcaacctcc acctcccggg ttcaagcaat tctcctgcct 131520
    cagcttcctg agtagctggg attacaggca tgtgcaacca cacctggcta atttttgtgt 131580
    ttttagtaga gaaggggttt caccatgttg gtcaggctgg tattgaactg ctgacctcat 131640
    gatccacctg cctcggcctc ccaaagtgct gggattgcag gcatgagcca ctgcacctgg 131700
    ctgaatgtct tgtttttgat taggcactta agaaaggcct aggtactaac cataaaatat 131760
    atttttatac cttttgttga tactatatat atagaaaact gcacttatca taaccttaga 131820
    caccttgaag aatgttcaca agcagaacta acccatgtga cccagcatcc agatcaaaaa 131880
    cagcattatc agcccctcta gaagccctct tgggcccctt ccattcactg tccttcttgt 131940
    caccagggta gctactatcc tgacttttga tggcatagat tagcattacc tgttcttgtc 132000
    attttataaa taaaaccata ctgtgtattc ttttcttgta cagctttatt gtgctaattc 132060
    acatttacat catacaattc agtggttttt atatggtcac agagttaggt aaccattacc 132120
    acatcgattt tagaacattt ttttcactcc agatagaaac cccctttact taaactccaa 132180
    atcccccact ccaccagccc taggcagcca ctagtctact ttttatctct atagagacaa 132240
    tagatttgct tattctggac atttcataaa catggaaccg tatattatgt ggtcttttgt 132300
    tgccaactgt ctttcactta gcatcatgtg ttcaaaagag catcatgtta tccatgtttg 132360
    gcatgtatca gaattttatt cctcattatg gccaaatatc ccattgcaag gatttatgac 132420
    attttatttg aattgtaccc tcctttctgc catttatcaa taatgctact gtgaccattt 132480
    gtgtacaagt ttttgtgtgg atacaggttt tctttttgtt tttaaatttg aggtggagtc 132540
    ttgctctgtc gcccaggctg gagtgcagtg gcacaatctc ggctcactgc aacctctgtc 132600
    tcctgggttc aagcagttct cctgcctcag cctcccgagt atctgggact ataggcacgc 132660
    accaccacgc ccagctaatt ttttagtaga gatggggttt caccatgttg gccagtctgg 132720
    tctcgaactc ttgacctcaa gtgatccacc catctcggcc tcccaaagtg ctgggattac 132780
    aggggtgagc cactatgccc ggctgtggtt ttcatttctt ttgttgtata tacataggag 132840
    tagaattgct gagtcaagag gtaactctta aacttattga aaaactgcca gattgttttc 132900
    cgaaaaggct gcaccatttt gcaatcccac cagcagtgta tgagttttac agcttctcca 132960
    catttcattg gaacttatta tctgtttggc tgtttttaaa aatgatagtc attccaataa 133020
    gttctacttc agtgtggttt ttgcacttct ctgatgagta atgatgttga gcatcttttc 133080
    atttgcttat tggcctttgt tctagctttg gaaaaatgtt tattcaaatc ctttggccat 133140
    ttttattttt atttttattt atttattttt ttttgagacc aagtctcact ctgtcagcca 133200
    ggctggagta caatggtgtg gtctcagctc actgcaacct ccgcctcctg tgttcaagtg 133260
    attctcctgc ctcagcctcc cgagtagctg ggattacatt tcaggcacct gccagcatgc 133320
    cgggctgatt tttgtatttt tactagtgac agggtttcac catgttagcc aggctggtca 133380
    caaactcctg acctcaggtg atctgcctgc ctaggcttcc caaagtgctg ggattacagg 133440
    cgtgagccat tgggcccagc ctagattttc ttttttcttt ttttttttga gaaggagtct 133500
    tgctcttgtt gcccaggctg gagtgcaatg gcacaatctt ggctcactgc aacctctgcc 133560
    tcctgggttc aagcgatttt cctgcctcag cctccccagt agctgggatt acaggtgcct 133620
    accaccacac ccagctaact tttgtatttt ttttagagac agggtttcac catgttggcc 133680
    aggctggtct caactcctga cctcaggtga tccacctgcc ttggcctccc gaagtgctgg 133740
    gattaccggc atgagctacc aggcccagcc aattttctca ttatattgcc caggctggtc 133800
    tcaaactcct gggttcaagt gatcctcctg ccttggcctc ccaaagtgtg gggagtacag 133860
    gcgtgagcca ccttgctcag cccctttgcc catttttaaa ttagattgcc tttttatatt 133920
    gagtttcagg agtcctttat atattctaga taaatgtccc ttatcaaatt atattatttc 133980
    caggtatttt cttcattctg tgagttgtct ttcctctacc ttttaaaaaa ggtgggtttt 134040
    tgtttgtttg tttgtttgtt tttttaagat aaggtctcat tctgctgccc aggctggagt 134100
    gcagtggcac aatcacagct cactgccacc tcaacttcct gggccgaagt gatcctctta 134160
    cttcagcctc ctgaatagct agggccatag atacacacta tcacacccag cttttttttt 134220
    ctgtttgtag agacagatct tactgtgttg cccaagttgg tctcaaactc taggctcaaa 134280
    gtgattctcc cacctctgcc tcccagagtg ctgggattac aggtgtgagc cacacgcaac 134340
    ctgtcttttc actattaata gtgtcttcct gcttcagcct cccgagtagc tgggattaca 134400
    ggcacccacc accatgcctg gctaattttt ttgcattttt agtagagaca gtgtttcacc 134460
    atgttcaccc ggctggtctt gaactcctga cctcaggtga ttcacctgcc atggcctccc 134520
    aaagtgctgg gattacaggc gtgagccact gcacccggcc aaaatattgc cttcttaaca 134580
    gtattgtctt ctaatttgtg aacatggatg tatcttcatg tatttatgtg ttctttcatt 134640
    tcagcagaat tttgtagttt tcagagtaga agcctttcac ctccttgggt catttattcc 134700
    tatgttttaa gttcttttcg attccattat aaatagaatt gttttcttaa tttcattttc 134760
    agattgtttg atgagagagc atagaaatac aagtgatttt tacatgttga tcttgcaact 134820
    tcaactttga taaatctgat tgttagctct aatagttttc ttgtggattc tttaggattt 134880
    tcaatatata agatcatgtc atttatggat agagatagtt ttttttctgg ctagaactta 134940
    cagagcaatg atgagtagaa gtggcagaag caaaaatctt tgtcttgttt cctatctgac 135000
    agggaaagct ttcagtttca tcatttaata tgatgttagg tgtgggtttt caataaatgc 135060
    cttttttcag attcaggaat ttccctatca ttcctgattt tttaaggctt tttttttttt 135120
    ttaaatcatg aaagggtgtt gaatattgtc atgttctttc tgtatcagta taaatgatcc 135180
    tatggatttt gggttttatt ctgttgatgt gaaatattaa ttgattttca gatgttaaac 135240
    caaccttgca tacctgagat gaatctcact tggtcatggt gtataatctt ttcaatatgc 135300
    tgctggattc catttactgg tattttgttg aagattttgt atctgaacgc ttaagataac 135360
    atttacactc tatcagaaat gaattgacca taaatgtgag agtgtatttg tgggttcttg 135420
    attctcttcc attccaaaga tagacataca tccgtctgta tgtctgtctt tatgccagta 135480
    ccatactctc ttgattacta ttgctttgta ataagttttg aaatcagaaa gtataaatga 135540
    gattttggta tctgagtaac agtcctcata gaattagttg ggaaatattc cctctttatt 135600
    ctggtccctc tttctttttt gtttaactgt gtatcttgga gattgttcct tctcaacaca 135660
    tgagagccgc tttccctacc ctcccacccc tgctatagag aggtctataa gtgtctgttc 135720
    aattatttta tttacttaac ctattactta gtcggggaca ttaagcttgt ttatgtcttt 135780
    tattttaaac aatgctgcag tgaataatct tgtatataag tcattttcca tcaatataag 135840
    tctctctgta actgaatttt tagaagtgga atttctaggt caacctatgg ctctgtattt 135900
    cacaaaaata ccaattctgg tttttcttgt ggaggtgggg agtaggaggt agaatgctgg 135960
    aggagaactt gctgtactca gctggctagt cattttagaa aggtttcctt agcttctttt 136020
    tgtcatatgg cctcaccaag aatcaaaaac attcctattt accctgtaaa catggggctt 136080
    tactacccaa gatacatatt tctggatgta tgacagcttt tcatattgaa gaaataatgc 136140
    tgtgagtaca gcacatttgt tggaacttag gtcgttaaga atgtcttata aattcataca 136200
    ttatacattt tattttattt tattttttag tttttgatac agagtcttcc tctgtcgccc 136260
    aggccagcgt gcagtggtac aatcttggct cactgcgacc tccatctcct gggctcaagt 136320
    gattctcatg tctcagcctc cagagtagct atggttacag gcatgcacca ccatgcccgg 136380
    ctaatttttt tatttttagt agaaactggg tttcaccata ttgaccatgc tggcctcgaa 136440
    ctcttggcct caagtgatcg gcctgcctca gcctcccaaa gtgctgggat ccttgtattg 136500
    ggtaaaagat gaatattgag ggctgcatgg tggctcatac ctgtaatccc agcactttct 136560
    gagactgagg tgggaggagt cctggagccc aggagggtga ggctgcagtg agttgtgatc 136620
    gcgccattgc acttcaacct aggaattata ggcttcagtc actgtgcccg gcatgtacat 136680
    tttaatattg tgctttcctc ttttagctat agtatgaggt tacatttcag agtcattgtt 136740
    gttaagcatc ttaatagtga tgaggttgag tgaaagttac ttctatttca aacactgaag 136800
    aaaattttgt acaaatctgt cacattccaa gcccaggact gattgtttca tatacttcta 136860
    attttacaat ttctattgta gtccagtgtg aaaaaagcca gtattaaaat actgaaaaat 136920
    tttgatgaag cgataattgt ggatgcggca agtctggatc cagaatcttt atatcaacgg 136980
    acatatgccg ggtaagctta gctcatgcct agaattttta caagtgtaaa taactttgca 137040
    tcttttaaat tttttaatta aattttacat ttttttctaa tctattatta tatgcccaga 137100
    actttcactt agagtgtgca gtataatgtg gtggttaagt ataaaggctc tggagtgact 137160
    tcctgggttt taatcttggc tctgccattt attggcagcc gctaacctct tggtatctca 137220
    gtttcttcat ctgtaaaatg agaataataa agtgaaaaga tgccaacatc atttactctg 137280
    ggctgcataa ctgatacttg gaaaaagtat tcctttgagt ttaagaatta agttggttat 137340
    tcattttagc ttgtaataaa aagatagtga ttcataggat atgccactta ctgaaattta 137400
    ccacagatcc aatcataaaa tcactttctc ttccctaaag atagcttgat taacatgtaa 137460
    aggtgtgtaa aggcttgatt acactaccct gatccgtacc ccagttccca gcagcaccat 137520
    gaaaaaggga tttcaacata tttaattact ttcagtagaa agtaacagtg gtaggccagg 137580
    cgcagtggct cacacctgta atcccagcac tttgggaggc cgaggtgggc ggatcacgag 137640
    gtcaggagat tgagaccatc ctggctaaca cgatgaaacc ccgtctctac taaaaataca 137700
    aaaaattagc cgggcatggt ggcaggcacc tgtagtccca gctacttggg aggctgagac 137760
    aggagaatgg cgtgagcccg ggaggcggag cttgcagtga gcttagattg tgccactgca 137820
    ctccagcctg cgcagtggag cgagactctt gtctcaaaaa aaaagaaagt aacagtggta 137880
    ttgggagact gaggagccta gaaagtactt gaaggaagta aaaggtttgt ttgaccacat 137940
    tgtatttgga aagccagctt tttcagctgt gtcagctttg tgtagtgatt tttagttctt 138000
    cttttagaaa ataacggaca aggccgggca cggtggctca cgcctgtaat cccaccactt 138060
    tgggaggccg agacgggcgg attacctgat ctcaggagtt cgagaccagc ctgggcaaca 138120
    tggtgaaacc ccgtctctac taaaatacaa aaagttagcc gggcgtggtg gcgtgtgcct 138180
    gtagtcccag ctactccgga ggctgaggca ggagaattgc ttgaacccgg gaggcggagg 138240
    ttgcagtgag ccaagatcac accattgcac tgcagcctgc gcgacagagt aagactctgt 138300
    ctcaaaaaat aataataaaa taaaaaagaa tggacagtaa acctaaatga gttcattccc 138360
    aaagatgatg ttattcttaa gggatggttc atttatttaa gaccttacat aaagtctatc 138420
    aattgcgtga tttttcactt ctgtaattgt gtgtatgtat aatgtaaata tatatgtttt 138480
    tgttttgttt tggttttttg agacggagtc tcgctctgtt gctcaggctg gaatgcagtg 138540
    gtgcaatctc agctctctgc aacctctgtc tcccaggttc aagcgtttct tctgcctcat 138600
    cctcccaagt agctgggact acaggcacgt gccaccacgc ccggctaatt ttttgtattt 138660
    ttagtagaga tggggtttca ccgtgttagc caggatggtc tcaatctcct gacctcgtga 138720
    tccacccgcc ttggcttccc aaagtgttgc tattacaggc atgagccacc acacccagca 138780
    tgtatttttt aaatgtataa aatgaagcag aaaagagaaa tgataatttt tcttcatctt 138840
    gaaagattat cttcaccagg cgcagtggct cacacttgta atcccagcac tttgggaggc 138900
    ctcggcaggc ggctcacttg agttcgaaac cagcctggcc gacatggtga aactccgtct 138960
    ctactaaaaa taaataaata aagatggttt taatatatgt tttagtttta tgattttagc 139020
    atctttctga aatttttctc aaggcaagta aatttgtatc agttggtata ttggtaccca 139080
    tctatgaaat aacttattag gaagatatct ctaaaataag atcactttgc ctaaaataaa 139140
    ctgatatatt gatgttcaca gaatttttct tttaaccgac ttgataaatg cattattctt 139200
    gacgtcaagt gatccacctt cctcagcctc ccaaagtgct gggattacac acatgagcca 139260
    ccgcacctgg cattattctt ataaaaggtt aaatttctag ttaagtttaa tgtcctcttt 139320
    gttcatgtac cattgcttat tttcttccct tcctactcac agtaatcatt cttatggtat 139380
    gcacttttgt ttgcttattt ttatgtaatt gatattacgc tccattctgt acgttgtact 139440
    ttcattcaca gtgagttttg gacattccta tgttcatcta tacagactta cttcatttta 139500
    actacactgt agtattccgt atgtaatatt tactataact catcactgta gcagagcatc 139560
    tcatagtgta tgtattactg ttttgccatt ttggtatcaa tgagtattta agtcatttgc 139620
    agtttttccc tcttataccc agtattacag aggatctctt tttatatgct tctttgtacc 139680
    aagaggcaga ttaaaaaatt tttttttgaa aaaatttttg aaaaaaaatg aaatgaagtc 139740
    tcactatgtt gcccaggctg gtctcaaact cctaggctca agcaatcctt ccatcttggc 139800
    ctcccaaagt gctggggtta caggcatgag ccaccatgcc tggcctacat tttaaatttt 139860
    gatagctctt acaatttact ttgtaaagta tctgcatcat tttatgttct caccagtctt 139920
    taataagaat acttcatact tttggctgga cacagtggct cacgcctgta atcccagcac 139980
    tttgggaggc cgaggcgggc agatcaagag atcgagacca ccctggccaa tatggtgaaa 140040
    ccctgtctct actaaaaata caaaaattag ctgggcgtgg tggcgcaccc gtagtcccag 140100
    ctactcgaga ggctgagaca ggagaatcac ttgaacccgg gaggtggagg ttgcagtgaa 140160
    cttagatcac accactgcac tccagcctag caacagagtg agactctgtc tcaaaaaaaa 140220
    aaaagaatac ttcagactta attttttttc cagtcttaag tgtttgctaa tgagattgag 140280
    tttcttttgg tatgtctctt gattgttcag gttttttctt ttatgaattg actgttcatc 140340
    tctttttcac attatttctg ttgggtgatt ttattagtga cttgttaaaa ttctgtatat 140400
    tttttcagca tgacacttca ttattcaaaa aaaaaaaaag attctctatg tttctcgata 140460
    ctaatcattg gttggtaata ccttaaaaat aagaccctta ctgtattttt tgcttttttt 140520
    tttttttttt tttttttttt tttgagatag agtcttgctc tgttgcccag gctggagtgc 140580
    aatggtatga tctcggctct cagctcactg caactgcaac ctctacctcc ctgtttcaag 140640
    caattctcct gccttagcct cccaagtagc tgggattaca ggcatccacc accacaccca 140700
    gctaattttt gtatttttag tagagacagg gtttcaccat gttggccagg ctggtctcaa 140760
    actactggcc tcaagtgatc cgcctgcctc ggcatcccaa agtactggga ttacaggcat 140820
    gagccacagt gcctagccac tttttgcttt ttaactttgt tttatagtac tatagtttta 140880
    gtataaacag atgtatgtat acacacaact atggctttat aatatgtttc agtcattgtt 140940
    agagcaaggc ctaccttttg ggtgcttctt ttacaaaatt gtcttggcta ttcttgtgcc 141000
    ttttttctta tttgtgaatt ttagaattgt gaattacctg ttgactcacc atgttttgta 141060
    aactgaggat tttgaatgga attgcactca attaaagatt atcttgcttt ctgtgcagca 141120
    atgttttatt tcaaataatc cctactttaa attacttagg atagctataa attgtgtttc 141180
    tggctttcta gatttagatg aaacgcttta aattgattgt tttctcctaa atttaaaact 141240
    gattgttaga agttaaagtc ttctgttcat tcttatttag gaagatgaca tttggaagag 141300
    tcagtgactt ggggcaattc atccgagaat ctgagcctga acctgatgta aggaaatcaa 141360
    aaggtttgtg gtgtttttat acttcatatt aagcctttac tcacattagt gattgactgt 141420
    aagtcaaaga ccacttaagg tttaaactgt ttattttgta aagtaaccac tgtatctttc 141480
    accttgtgtt tatagtcaga agtaagtaca agggcttcct gtagtcacat ctttatgcaa 141540
    tctcctctga atcaaaagtt agtgaacttg ctttgccact ccagaaggca catgaatatg 141600
    aaaaagcatt gtctattttc ttatttaatg gcaaaatacc cgacctaagt tggacttaat 141660
    gtttgagacc gtttatttta ttaaattata ttttttctct tttctttttt ttttttgaga 141720
    cagttcttgc tctgtcaccc agaccggagt gcagtggtct gaccgcacct cactgcaacc 141780
    tctgcttcct aggttcaagc gattttcctg cctcatcctc ctgagtagct gggactacaa 141840
    gtgcgcacca ccacacctgg ctaatttttg tatttttagc agagatgagg tttcaccacg 141900
    ttggctaggc tggtctcata ctcctgacct caagcaatcc atccgccttg gcttcccaaa 141960
    gtgctgggat tacaagtgtg agccaccatg cctggcctta ttaaattatt tttattaaat 142020
    ttcctcaaga ttgatgaaag taatgaaata taaaagtaat gaaatatatg tggaaaatag 142080
    actggattaa gaaaatgtgg cacatataca ccatggatac tatgcagcca taaaaaagga 142140
    tgagttcatg tcctttgtag ggacatggat gaagctggaa accatcattc tgagcaaact 142200
    gtctcaagga tagaaaacca aacaccgcat gctctcactc ataggtggga attgaacaat 142260
    gagaacactt ggacacaggg tggggaacat cacacgctgg ggcctgtcgt ggggtggggg 142320
    gctgggggag gaatagcatt aggagatata cctaatataa atgacgagtt aatgggtgca 142380
    gcacaccaac atggtacatg tatacatatg taacaaagct gcacgttgtg cacatgtacc 142440
    ctagaactta aagtataata aatttaaaaa aaataaatat atgtggaaaa tattaatagg 142500
    tcaaaattca aattgttcat ttaatcagaa gagtagttta gtcaaatcca agggttagac 142560
    aacagaaatc ttttttgtca agtgcattct ttgtgactga tttcattttc ttcctggttt 142620
    acacaggaag atttcagaaa caaatgtgga tccgtgacag atggtatcta gaagttttta 142680
    gtttggttga attgacagta ttttattgag taaaagatac taatttttgt aagaagaaaa 142740
    attcaatttt gataagtatg tttaagatta agagctattg gccaggcgct gtggctcatg 142800
    cctgtaatcc tagcactttg ggaagctgga gcaggtgggt cacgaggtca agagattgag 142860
    accatcctgg ccaacatggt gaaaccctgt ctctactaaa ttagccaggc gtggtggcac 142920
    atgcctgtgc acccgcctcc gggtttaagc gatcctactg cctcaggctc ctgagtagct 142980
    gggattacag gcgccatggc taatttttgc atttttagta gagacagggt ttcactacat 143040
    tggccaggct ggtctggtct caaactcctg acctcaggtg atctgcccgc cttagcctcc 143100
    caaagtgctg ggattacagg catgattcac catgtctggc catttatctt attttctttt 143160
    tttttttttt ttttgtttga gacggagtct tgctgtgtcg cccagagctg gagtgcaatg 143220
    gtgcgatctc agctcactgc aacctctgcc tcctgggttc aagcaattct cctgcctcag 143280
    tcttccaagt agctgggatt acaggcgcgt gccaccacat ctagctaatt tttgtatttt 143340
    tagtagagac agggtttcac catgttggcc aggctggtct cggaactcct gacctcgtaa 143400
    tctgcccacc tcggcctccc aaagtgctga gattacaagt gtgagccact gtgcccagcc 143460
    atcttatttt ctttcttttt ttttgtcggg tgggaggggg acagagtcta gctctgtcgc 143520
    caggcttggc tcactgcaac ctctgccccc caggttctag caattattct gcctcagcct 143580
    cccaagtagc tgggattata ggcacctgcc accacgcctg gctaattttt tgttattttt 143640
    agtagagatg gggttttgct atgttgacca tgctggcctc aagtgatccg cccaccttgg 143700
    cctcccaaag tactgggctt acaggcgtga gcttgtattg ggtaaaagaa caatattggg 143760
    ggctgcatgg tggttcatac ctgtaatctg agcactttgt gagactgaga tggaaggagt 143820
    gttggagccc aggagggtga ggctgcggct gcagtgaatt gtgatcacgc cattgcactt 143880
    ccacctaggt aatggagcaa gaccatgtct ctaaaaaaca aaacacaatt tttttaagga 143940
    atactgggaa gaggtcagtg gtggttttag aacagaggaa gtgccagatg acctttgtga 144000
    ggcattggcc aggaagaact ctacagtgtc tttaggtagc ttctgtccat aaggataatg 144060
    gggtctcctc cccagtatta atagaaaatc tctgagctgt ttttttttgt ttgtttgttt 144120
    tgtttttttt tcctgagatg gagtctctct ctgtcggcca ggctggagtg ctgtggcgcg 144180
    atcttggctc actgcaagct ctgcctccca ggttcacacc attctcctgc ctcagcctcc 144240
    caagtagctg ggactacagg tgtccaccac cacgcccagc taattttttg ttatttttag 144300
    tagagatggg gtttcaccat gtcagccagg atggtctcga tctcctgacc tcgtgatccg 144360
    ctcgcctctg ccttgcaaag tgctggagtt acaggcgtga gccaccgtgc ctggcctggt 144420
    ttttttgttg ttgttattta tttatttatt tatttatttt ttgagacaga ctctcgctct 144480
    gtcgcccggg ctggagtgta gtggcacgat gtcggctcac tgcaagctct gcctgccagg 144540
    ttcaagccat tctcctgcct cagcctcctg agtagcaggg accacaggcg ctcgccacca 144600
    cgcccggcta attttttgta tttttagaag agacggggtt tcaccgcatt agccaggatg 144660
    gtctcgatct cctgatgtcg tgatccgccc acctcggcct cccaaagtgc tgggattaca 144720
    ggtgtgagcc accgtgcctg gcctgatttt tttttttttt taatctggtc tcatacctct 144780
    gacagctcat gaagaagtgc tcctgcttca tatgtatatg tgttagcata gtgttaacat 144840
    agcataggtg ttcggtgttt gcagtttctg tttgttttat atgaattaag gtgtattatg 144900
    agcagttgaa gatatatagg aaattttttc ccaaaccact atctctgctc gttctattca 144960
    ttcagtctgt ttatgttatt ccttcattca ttcattttat agaacagtgg agtgcctact 145020
    gtatgcatct attgttctgg gtcctgggga agaaaacaaa gttcctgctt tcatggaact 145080
    tacattatat tggcggagac agtaacagac aaacaaatgt agcctgtgta catgtgttac 145140
    atgaaaagca gggtaggggg ctgggagaga gtagtaggga gtgctatttt cgaggtggtt 145200
    gtcaggaaag gcctcactga ggaggtggca ttttgagtag acctgagcgc agcgggggcg 145260
    taagcccagg cagcatgtgg aggaagagtg ttcttggtga aaggaacaag gatagaggcc 145320
    cgaagctaga gagctcagca tgatcaagga acagcaagcc ccgtgtggct ggaatggagt 145380
    gagcaaagga atgagcagta gaaggtgagt gagttgggag gtcaccagag accatggcaa 145440
    ggacttgaaa gtgtcaggga cacattggaa gttggagcag ggaaatgatg ggatttatgt 145500
    tttgtttttg ttttatgttt agtgttttta agggattgct ctatcagcta tttggaaaat 145560
    ttagtgtagg gcttcaagaa gagaagcaga gaaacaacat tcttgccata gtcatagtct 145620
    aagtaaggga tgatggtggt gtggattagg ctggtagtgg aagaccagtc cagttcgggt 145680
    tgtatttgaa ggtagaggca aaaagattat atttctacca gcaagcccat ctatgaagtt 145740
    acttgtatta ttaatttaat tgagacatgc ccacataaac taataaatag gaatttctgc 145800
    agtttggtta aacacccctg tatatcctgg ttcttctttt agttgtccag atgtctcttt 145860
    aagtcaagta ttttttggtg gtgtaggagc ctagagattg aatttattca cccaaaaggc 145920
    atttgagtga ttactatgtg ccaggcacta tgctgaatgc caaggatgta aataagaggg 145980
    cgtagtctca gtctgtttta ctccagcttg gttccttttt aatgaccctg acttgttaag 146040
    catatcagtt atcctacaga atgtttaatc ttctgtactt tcctggttgt gttatttagc 146100
    ttatttctct ttccttgaca tttcttgtaa actggaagtt acacctatag tcttgatgat 146160
    tcgtgttaca cattttagat tagaacacat catgtgttgt atatggtgtt tttgaaagcc 146220
    tctctgtata ttggtctgta cattaaaatg ttgcctgaat ggatacacat aaaatttaac 146280
    agtgattaca ttagagatga gaagaaagag gtgcctttta cttttcaata taccttttcc 146340
    tctgcttttt gaactttctt gccctatgca tacgttattg cttaatcatc cacctcatct 146400
    cttcccctgt ggctttctgt tgcatttgga atgaaatcta gcctctttgc tgttacctgt 146460
    ggatgtccct tgctggcctc tatcacctta ctttgaacca ctcctttcat ggactgagct 146520
    ctcattggac tatcttttat tcttttgctg aagtttcttc actttgagtg cctctgcagt 146580
    tgctatttca tggctgtggc aagccctgcc atggctttca tgcaaggatg gttcctcctt 146640
    ctcatctcaa tattatctct tcagagaggg accttcccaa ctccgatgat ctaaaatcct 146700
    ttgtatatac cactcactac cacttctttc ttttcttttc cttttatctt tttttttttt 146760
    tttttttttt gagatagggt cttgctctgt tgcccaggct ggaatcacga ctcactgcag 146820
    cctcatcttc ttgggctcaa atgatcctct cacctcagcc tctcgagtag ctggaactgc 146880
    aggcacacac caccatactt ggcttattat tttacttttt gtagagacag ggtttcacca 146940
    aggctggtct caagctcctg ccgcaagcaa tccacatctc tcagcctccc aaagtattgg 147000
    gattatagga gtgagccact actcctggcc tattttctta ttcactgtct aaaattatct 147060
    tgttcattta tttacatact tgtttatagc ttatttctca gctggacatg gtgcctcaca 147120
    cctgtaatct caatactttg ggaggctggg ttggagaatt ggttgagccc aggacttcaa 147180
    gaccagcctg ggcaacaaag tgagaccctg tctataaaaa attgtttaaa aattagctgg 147240
    gcatggtggc acatgcctgt ggtcccagct acttgggagg cagaggtggg agaatcgctt 147300
    gggcccagga ggttgaggcg acggtgagcc atgattgtgc cactgcactc tagcctagtg 147360
    acagagtgag accatgtgtc taaaaagtaa ataaaaatag tttctctttc atgactagaa 147420
    tattacctct atgtgggcag ggagtttgtc tatactattt ggcactatat ttcctgattc 147480
    tgaaattatg cctagcacat ggtaagtact ccttaaatat ttattgactg aattatttaa 147540
    tacttaagaa tttcatttgg gattatctga gtggtaagat tacggattat atttatgtaa 147600
    gaaaaaatca ttttttaaac ttggttgccc tttgccacac tgacatagac actaagtttt 147660
    cttagccaga ttacttccga ggatactcac agaggccatt ctcttctcaa tccccaaata 147720
    attgatattt cttagcactt tcaagctaat gcaattctta gatgatgtat ctgtgtatat 147780
    catatcctca ttctacaaat gtagaaattg aagtctgggc acagtggctc tcacctgtaa 147840
    tctcagcagt ttgggaggcc aaggcgagcg gatcactgag gacaagagtt aagaccagcc 147900
    tggccaacat ggtaaagcct tgcctctatt aaaaatacaa caattagggc cgggcgtggt 147960
    ggctcacgcc tataatccca gcacgttggg aggccaaggc aggcagatca cgaggtcagg 148020
    agttcgagac catcctggct aacacagtga aaccccatct ctactaaaaa tacaaaaaat 148080
    tagccaggca tggtggcacg cgcttgtagt cccagctatc gggaggctga ggcaggtgaa 148140
    tcccttgaac ccgggaggcg gaggttgcaa tgagctgaga ttgcaccgct gaactccagc 148200
    ctggtcaaca gagggagact ctgtctcaaa aaaaaaaaaa aaaaacaatt agccaggcgt 148260
    ggtggcgggt acgagtacct gtaatcccag ctactaggga ggctgaggga ggagaatcac 148320
    ttaaacccag gaggtggagt ttgcagcggg ctgataatgc accactacat tccagcctgg 148380
    gcaacagagt gagactctgt cttaaaaaaa aaaaaaagaa agaaagaaat tgaggaatgt 148440
    ggagattgtg gtctgtgatt tgttaggaat cacacagcag gttagtagca actacagggc 148500
    tttggttcag aataccacct tgacaatggt ttgtttacag ttcggctccc cttcctctgc 148560
    ctttctctcc ttccttattg agggcagctg gaaagaattt tcatcattta ctagcctata 148620
    gctttaattt gagttttgaa accttgataa tagagcacag aggaaaagac tgagttttct 148680
    ttttttgaga cagtcttgct ctatggccca ggctggagtg cagtgacacc atctcagctg 148740
    gttgcaacct ctgcctccca ggttcaagca attctgcctc agcctctcga gtagctgaga 148800
    ttacaggcac gtgtcaccac gcccagctaa ttttctgttt ttgtttcgtt ttgttttttt 148860
    ctgagatgga gtcttgctct gtcacccagg ctggagtgca gtggtgcgat gttggctcac 148920
    tcaaacctct gtctcctggg ttcaagcaat tcttctgcct cagcctcccc agtagctggg 148980
    actacaggta cgtgccacca tccctagttc atttttgtat gtttagtaga gatggggttt 149040
    cactatgttg accaggctgg tctcgaactc ctgatctcag gtgatctact cgtctcagtt 149100
    tcccaaagtg ctgggattat tggcacacgc ctatttttgt atttttagta gagacggggt 149160
    ttcaccatgt tggttagact ggtctcaaac ttctgacctc aagtgatttg cccgccccag 149220
    cctcccaaag tgctgggatt acaggcgtga gccaccgtgc ccagccaaga ttgagttttg 149280
    aaaagagcct tctgagatta tgagaagggc aagcaagata acttaagaag ttacattaaa 149340
    atcatctaag agacagtgta acaagaagga attgtaaaat gatgttatga gcacgtgccc 149400
    aatgtagtgg caatcccttg tgcttcgata cattggtggg agacaaaact gtacttaaat 149460
    tgataaatcc cttacatgtc attttaagga gcttagactg actcccatca tgtagacatc 149520
    agagatttct tttttttttt tttttttttt tttttttttt tttgtgacag agttttgctc 149580
    ttgttgccga ggctggagtg caatggcgtg atctcggctc accacaacct ccacctccca 149640
    ggttcaagca attctcctgc ctcagcctcc cgagtagctg ggattacagc catgcaccac 149700
    cacgcctggc taattttgta tttttagtag agacggggtt tctccatgtt gtggctggtc 149760
    tcgaactcct gacctcaggt gatcctcccg cctcagccac ccaaagttct gaaattacag 149820
    gcgtgagcca ccgcgcccag cccagagatt tctaaacaga gttctaacca gatgcttttc 149880
    cctgtcagta gaatgagaat gaattggagg tgggagagac tggcatgagg gacaccagtc 149940
    agccagtgga attagctggt aatgttgata ggagaagaaa aagattcaaa gttaggtagt 150000
    ggtagcaaga attagaggga aggtcggatt tatgatatgt ccaaggttga attctaaggt 150060
    gaaatttggt ggcagatttc atgtgtaaat tgggaaggta gattgagttt ttttaacatg 150120
    ggttttctaa catgtcaata gagtgactct gcaggggggc ctgacgagag aacagtgcat 150180
    ggggtgattc aacagccagt tgagccttca tgcagagcat ttaacactgt gactctgtag 150240
    actctggttg gcagtaaaat ttcattaaac caatatttaa acccttaggt aataataaaa 150300
    attgagggaa aaggatccag gttttgtatt ttttatgaat tcagttattg aattaaacag 150360
    gaccttgcct caagaaataa tctaccaaca attaacttgt tttaaagcaa agttaggaag 150420
    tgagcatgtt caaattatta aataaaaaag taagctgtgt atttcattca tagaaataga 150480
    ggctggccta cttcggatga ttctcagcat gtgattacag atgtgggctt atacatccta 150540
    gggagttaag gcgtactctg gcttggatag agtagagctc tttgaaactc ttctctcacc 150600
    cagctagttt atatagacta gagaactaga atgtagcagc atactctgtc ttagaagccc 150660
    ttttatatag gagctggtct ggaaggtttg aaaacataac aaatgtgttg gtgtctccca 150720
    atgtattgct agattcttac ccaagagcat tatcctggtt agggtttggt ttggttttgt 150780
    tttgtttttt aatgtttgcc acaaactaac actagatgtt agttctttca tcaagtgagg 150840
    agagtagaag aaaagtccag aactctgaaa caccttttca aaagtttttc aagccatgat 150900
    gtttgcaagt taaatgctct gttatgtaag caatataatc agtttttatt aatgtaacat 150960
    tccttagtgt tttggggtat cacacaaaaa agaatatcca tatctggaag caacagcttt 151020
    taaataagag cattgtggtg gtggtggtga tagtggtttt tttttttttt tttgagttgg 151080
    agtctcgctc tgttgcccag gttggagtgc agtggcacga tctcagctcg cttcaacctc 151140
    tgctcccagg ttcaagcaat tcttctgcct cagcctcctg agtagctggg attataggca 151200
    cctgctacca tgcctggctg atttttatta ttttagtaga gacaggtttc accatgttgg 151260
    ccaggctggt cttgaactct taacctcagg tgaatcaccc acctcggcct cccaaagtgc 151320
    tggaattaca ggcatgaacc accatggcca gccaaataag agcattttta atgtaaaatt 151380
    atgcatgaaa tgtacattca attttgtctt tgtttactag gatccatgtt ctcacaagct 151440
    atgaagaaat gggtgcaagg aaatactgat gaggtaaatc ctacctttag gataaaaaga 151500
    tttctgttta taagtgccac cctcatgtaa gtgaggttta aaattttcct tttctttagg 151560
    tcccatgttt aagcagcatg gcacatttat gttctcttac ccagaatgta ccaagaaagg 151620
    gtggtccctt cttaacatct aacaattgcc tggtagtagc agtgaaggta tcttcagtca 151680
    gaggctagga ccactgaagg atatacatgc attcaagttt ccatcagcca gcaggcatca 151740
    gtaatcagtg tgtagatcaa aagctcaaat gtttccttcc ccactggcag ttttacttca 151800
    agtagtggag gcttgctttt ttaatagtta attaagtaca ttgagagatg ggaggtgaaa 151860
    aaaggaaaat gttttatttt gaccatctaa tatgaaagta gttcggtgtt aggtatccag 151920
    tagttgacac tggaagacag ggaatgacat gttaatattc atagccagag ggtggcccag 151980
    gttttttcgt acatgggaat gaaattctta tccaaataag tagaaattat gtgcgtaagc 152040
    catttgttaa gagcactgag tatgtgcatc tcgatccatc taatgaataa ccattatcac 152100
    cagtttaaat tattttcttt aggcccagga agagctagct tggaagattg ctaaaatgat 152160
    agtcagtgac attatgcagc aggctcagta tgatcaaccg ttagagaaat ctacaaaggt 152220
    aaggatgact tcgttttgtg taaactaaaa agtattattt tccaggtgta aaaataaaaa 152280
    agaacataag gggtttcttt gcctttgaag gattaactgc tgtggggatt accttcttat 152340
    cataagcaac tagaaaattg acaaactaaa tgaaacaact gtttgcatat attggacaat 152400
    gggcaataca gggaaaccat ggaaaccaaa cagagcccag tagtcttgct gaacgaaaga 152460
    gttaaatatc aaagttcagg ccaggtgcag tggctcacgc ctgtaatccc agcactttgg 152520
    gaggccaagg cgggtgaatc acttgaggtc aggagttcaa gaccagcctg gccaacatgg 152580
    tgaaaccctg tcttagccgg gtgtggtggc aggcacctgt aatcccaact atttgggagg 152640
    ctgaggcagg agaatcgctt gaaccaggga ggcggaggtt gcagtgagcc gagatcacac 152700
    cactgcactc cagcctgggc gacgagcgaa accccatttc aaaaaaaaaa tcaaagttca 152760
    gagagctcaa tttgagtaga agttgtagga taaggtagca gaaaagagga agctgcccag 152820
    aaagaaagcc gtagagatat ttagagagat tcccatggat ccttggccta ggagtgatct 152880
    gtatatgtgt ggggtgaaaa cgcatgtgtc caggtagaga accccccaga aattagtagg 152940
    ctgaatgatt gctggaacat agggctaaga aaagttcatg gccagaagga tctggccaga 153000
    gtagagagac ttagtaatac acaaggcatt gggtagtgtc ttcacagagg ttatgcctta 153060
    ctactgaaga taaattagtc ctagagtaca agcacctgaa ccaagtttca aagcaaattt 153120
    ttaaagggtc aaattaccta acaactgcat gccaaaacaa aggcctaacc ctctttacag 153180
    taacacaaca aaattcagca cttcacagtg taaagttaga atgtctgacg tccaggctgg 153240
    gcgcagtggc tcatgcctgt aatcccagca ctttgggagg ccgaggcagg tagatgacct 153300
    gaggtcagga gttcaagacc agcctggcta acatggtgca accccgtctc tattaaaaat 153360
    acaaaaactt agccaggcat ggtggccggc acctgtgatc ccggctactt gggaggctga 153420
    ggcaggagaa ttgcctgaac ccaggaggtg aaggttgcag tgagccgaga tcgcaccact 153480
    gcactctggt ctgggcaaaa agagcaaaac tcaggctcaa aaaaaaaaaa gaatgtctga 153540
    cgtcaatcac aaattaccaa gcatgacatg aagttgacct ataaccagga gaaaactcaa 153600
    tctatagaaa cagacccaga tgtgagaaag atgatgaatt tagcagacaa agaccatcaa 153660
    gtggctattt taaatattaa aaatatgttc aagtggccag gtgcagtggc tcatgcctgt 153720
    aatcccagca ctttgggagg ccaaggtggg taggagttca agaccagctt ggccaatatg 153780
    gtgaaacccc ttctctacta aaaatacaaa aaaattagct gggcatggtg gcaggtgcct 153840
    atagtcccag ctatatggga ggctgaggca caagaatcac ttgaacccgg gaggtggagg 153900
    ttgaggttgc agtaagccga gattgtgcca cttgtactcc agcctggaca acagagtgag 153960
    actctgtctc aaaaaaaaaa aaaaaaaagt taaagaaaac aagagtataa tgagaaaaat 154020
    gcaaaatagt tttaaaagaa ccaaatggaa tttcttaaaa taaaaaatac cagaaatggg 154080
    ggccgggcgt ggtagctcac gtctataatc ccagcacttt gtgggggctg aggcaggcag 154140
    atcacctgag atcggtagtt caaggccagc ctgaccaaca tggagaaacc tcatctctac 154200
    taaaaataca aaattagctg ggcgtggtgg cgcattgcct gtaatcccag ctacttggga 154260
    ggctgaggca ggagaattgc ttgaacccgg gaggcagagg ttgcggtgag ctgagattgc 154320
    accagtgcac tccagcttgg gccacaagag tgaaactccg tctcaaaaaa aaaacaaaaa 154380
    aaaacagtag actcgaagaa ctagctgagt ttttctttac tttaggcagt aagtgtgacc 154440
    ttttgcaggt gactacttta gttcctcatg tcctcattag tagatcagag aaattcgaca 154500
    ccaaaacccc aaaagaaaaa ccccttctaa tcctcattcc atgattttat gaatgcatga 154560
    agtcctaggc ctgcgaagga atactcattc tctttatcct gtgttgatac ctctctgctt 154620
    caacctccaa ctcgacattt gcctatagga tgtacttgga cattcagcat aaactacctc 154680
    acaccattac tgaattgctt catgtgcaca tgtcccatgc cacaataccg gggaccttgt 154740
    cttccgtgat atttgtccgc agtgctgtga ctacaggagg gagtcagtga atgtctgcat 154800
    gtgtgtcttt accatccctc ttgaatatgc tctagggtta attcctagaa gtagaattac 154860
    tctattgaaa attggcaata tttttcattc taatatctat tgccaacatg ggaaagcaag 154920
    tctggatgcc agtccttgtt atatgcccct tgggtaagtt acgtaacctc tttaagcttc 154980
    tgttcactca tattttaaca aggaaaatta caatatttta cctcacaaaa ttgtagtcag 155040
    cttctggctg tcttaaactc tggtatatag taaacactaa gtgttggtgt ccatccttaa 155100
    tttgtaataa taggtcactt gttagagaaa tgcaccttac cattttcttt tcttttcttt 155160
    tttcagttat gactcaaaac ttgagataaa ggaaatctgc ttgtgaaaaa taagagaact 155220
    tttttccctt ggttggattc ttcaacacag ccaatgaaaa cagcactata tttctgatct 155280
    gtcactgttg tttccaggag agaatgggag acaatcctag acttccacca taatgcagtt 155340
    acctgtaggc ataattgatg cacatgatgt tcacacagtg agagtcttaa agatacaaaa 155400
    tggtattgtt tacattacta gaaaattatt agttttccaa tggcaataac ccatttatga 155460
    gagtgtttta gcctactgga atagacaggg accacatcct ctgggaagca gataagcata 155520
    gaactgatac ttgatgcaca ctcgtagtgg taactcatcc ctaatcagca ttgtaaagca 155580
    ggtgccagag gtggtttgct ttgtccttcc aaagcaggtg agtcagcccc accgagagcc 155640
    aggcagcttt gagtggcagc gtggtgctag cagcttcagc ggaacagggt gagagttaat 155700
    tatgcagtct tcttgacagc ggcattaatt tggaaggaaa ctgacaagtc atgggtcaag 155760
    tttcagtgac ttcctccttc ctctgatggc agtatatagt tttcacattt taattcctcc 155820
    tcctgagatg cactatactt aaaaccattc tctcccctgc taacagaagg gtgtgaatct 155880
    ggtttacttt gagcattagg atttgcccct ttggaattct gcactccagt tacttaactt 155940
    tcccttcaga atacatgtgg aaagaaagaa agaaatagcg atgactccac ttttgcccct 156000
    gtggcacctt gaacaaagca gttcttccca aattatactt tttttttttt taaataaggt 156060
    gagcaggatg actggggaga gagaaacatt tgactttgac tgcctccccc attctttgct 156120
    gtgagctgga aagtgtgcag ttggtcgtct ttcttctcct ttctttagga tagtaagaga 156180
    ctcactcact gcacttctgc tcagttggct tctgcatcgg gatcacacag ccatcagcag 156240
    gactgcccag ttggtgagca cactccattg accacgcggc gccagcgctt cctcaatgca 156300
    catgattgag aggaaagaaa gttctcttag atgttactgc ttttgctcag actttgcaaa 156360
    aaaaaaaata tatatatata tgtataaata tataattatt aatcactttt gtccttgaga 156420
    aagtcttgaa tgaacagaga atttattcca ttgcaatatt tgattgtata gaggcacact 156480
    gtttcatcga cagaagaagc aaaaaggctt tgtgtaagtt tttggtacta tgtaccacct 156540
    ctgttattct tttaaagctg aagtattcat gtacttaaac catattatat ttaattgtgt 156600
    ttgattttaa aatatatata tatgaattct atttaaaatt gtgtcaactt tctgctttca 156660
    gggcatttat ggctcttctg ttgaaatata ttgatctttc caaatatttt catttgcttt 156720
    ctaaaaaccc agaacatgag ccactactgg actttgcctt gtgtttgaag tgtatggcat 156780
    aaacccaagg tttttattag tcatctatgc tgtgattaat tcattttgtt cttttaacaa 156840
    aatatttcca tccacttcac attgcttcaa tctttaacag aaaagcaata taaaggttat 156900
    agaataaaat gtggttttgg gcaactcttg ctgcctctgc atgttttgga ataacaattt 156960
    ctacaagact ctaggctgtt taaactagtg ctttcagtta agataaattc taatcatttc 157020
    tttgtatata cattttgtgc ttctgagcta gagatgccaa gtagttgtaa actgcttata 157080
    aagagaatag cagcaaattt gagactcggc tacttttttc tgccccacct gctttgagac 157140
    acagaagcgg agtgtggccc gaaattatta gccagattta atatttgatc taaagtaggt 157200
    ccttgtactc attttaaagt tggaatttga ttcctccaac attgagcacc caccatgttc 157260
    caggctctgt gcattgtgcc cacaaaataa gattccctgg tggagttttt atgggttcaa 157320
    ataatcagtt gaacaccctt catctttatc atgttgttga cattgacaca aattgtttaa 157380
    aaagaaaaga tattagagag aaagtggtac ctttgtaact tgatgtgtct tcatcattcg 157440
    gtaagatttg atgaaagtaa aaagcaaatg tcagccaaat ccagtgaaca gcaataaaac 157500
    agggagtaac tttttataac tttttctact tggatttcaa cattcagtag agcttttcga 157560
    aatgtaagta gtttacagta ctggaggttt gactagttca gtaggaattt ggaggggaag 157620
    gtcattctga attgtaacaa agtacaaact tctttgctgt tttatttaag tactgagagc 157680
    taagcacctg atgaagtgac tgacctctct ccagtgacag tgtttgggta cctgcctgac 157740
    ttcaggagtg gggtttatgt ttctacacag tgaccttttc tctcgccctc tcctccctct 157800
    tgcccacaca ccagttgatt ggacctgggt tgaactcctg atccagacag gcccaagaca 157860
    gttcttaatg ttaagaattt tggggccggg cacggtggct catgcctgta attgcaacac 157920
    tttgggaggc cgagacaggc ggatcacttg aggtcagggg ttcgaggcca gcctggccaa 157980
    catggtgaaa ccctgtcttt actaaaaata caaaaattag ctgggcatgg tggcgcacgc 158040
    ctgtaatccc agctacgtgg gtggctgaga caggggaatc gcttgaacct ggaggcggag 158100
    gttgtgcaat gagccgagac cgtgtcactg cattccagcc tgggtgacag agggagactc 158160
    tgtctccaaa aataaaaata agaaaaagaa ttttgggcta ggtgcagtgg ctcacgcctg 158220
    taattacagc attttggaag gcccaagatg ggcagatcac ttgaggacag gagttcgaga 158280
    ccagcctgga caacatggtg aaactccatc tctactaaaa agacaaaagt tagccagatg 158340
    tggtgatggg cacctataat cctagctcct cgggaggctg gggcaggaga atcacttgaa 158400
    cccaggaagc agagattgca gtgagccaag atcacatctc tgcactccag cctgggcaac 158460
    agagcaagac tctgtctcaa aaaaaaaaga atttggccag gcgcagtggt tcacgcctgt 158520
    aatcccagca ctttgggagg ccaaggcagg cagatcacga ggtcaggaga tcgagattgt 158580
    cctggctaac atggtgaaac cctgtctcta ctaaaaatac aaaacattag ccgggtgtgg 158640
    tggtgggcac ctgtagtccc agctactagg gaggctgagg cagaggaagg atgtgaaccc 158700
    aggaggcgga gcttgcagta agccaagatc gtgccactgc actacagtct gggcgacaga 158760
    gtgagactcc gtctcaaaaa aaaaaagaat tttggccggg tgcggtggca catgcctgta 158820
    gtcccagcac tttgggagac caaagtgggc ggattacctg aggtcaggag ttcaagacca 158880
    gtccggccaa tatggcgaaa ccctgtctct tactaaaaaa aatacaaaaa ttagccaggt 158940
    gtggtggcgg gcacctgggg aggctgaggc agggagaaat gcttgaaccg gggaggcaga 159000
    ggttgcagta agccaagatc gtgccactgc actccagagc aagactcttt ctcaaaaaaa 159060
    aaaaaaaaag aattttgcat ggggaaggag agatactgtt caccatctgg aatggtgctt 159120
    ggatgtggca cttacaaaat caggagccag cactgcatgg acaaacagaa gcatgtgggc 159180
    ctgagatagc aggtaccttg ataaccctga agacatcctt ggtttctgca tctattcctg 159240
    catccttgca ttggactaca ttaatctgtc agttatcctt ataatgattt ttgatttttt 159300
    ttttttgaga tggagtttcg ctcttgttgc ccaggctgga gtgcaatggc acgatctcgg 159360
    ctcaccacaa cctccacctc ccaggttcaa gtgattctgc tgcctcagcc tcctgagtaa 159420
    ctgggattac aggcatgcgc caccacacct ggctaatttt gtatttttag tagagacggg 159480
    gtttctccat gttggtcagg ctggtctcga actcccaacc tcaggtgatc accctgtctc 159540
    ggcctcccaa agtgctggga ttacaggcgt aagccatggt acccggtctg ttttttgatt 159600
    ttttgaaacc agtctgaagt gagttttttt aattacgtga aaggagtttg gctaaaatac 159660
    tgccatactg ccctaatgcc taatgattat gtattctcag catgtctgca aagtactgct 159720
    gatttctgga gaataatttt tctttagtaa acttcactta agtcgtcatg tgtattctct 159780
    caaaatggta tcctaaccta atggagctaa aagacacccc ttgtttttat aacaagcagt 159840
    tactgaggcc caggaagggg agaagtccct ggcttgtgag atgatcacca ttagaactca 159900
    ggcctgggcc agtgcctttt catgcttctc agatccttcc aaagaataat gaagattata 159960
    accgctttta gcaattgtaa taaacccaga aatagaaagc tttttggtta gagtactggt 160020
    agaagtttgg cgggagagat aatttttaca aaatttgtaa atacctgcca attctatata 160080
    ctaggcaagg tctctggcct tgtaaaaccc ctcaaggtta caactttggt ggcccacact 160140
    aatagttacc cactgaggcc ctctccgggt gaacattgag cactagagga agcccctctg 160200
    cttgggcagg actgggcgtg gtgcagagta ggagcggtga tactgtggat tctgggcagg 160260
    tggagatggc cagtgatgtc caataaagga cactggaggg agcagtgtga gtaaaggccc 160320
    tgagggcatt catgttcagg gagggttgct gcccactggc ttgcttggca cacaggagag 160380
    tgggtattcc tgccttagta actttatgta aacaagtatt tcctcagtct gttcctctca 160440
    aactgcctgc tctggcacat tcagaatgtc acagaactca cctggatgca ttcagcccct 160500
    tgcctaaagg tgacagtgca tctccttccc caccccaccc ctcataccac tgaagcacct 160560
    gtcagactgg cccagtctgt gggcaaggag cctagagagg gcttagtttc agcttgaaag 160620
    gagctgggat ttaccaagaa gcaaatgaga gacgaggatt gcaacaactg tgccatttcc 160680
    ccagcttcag ctgactcctg tatattgact gtgccttcag actcatccgt aagtgacccc 160740
    aggctggcct ctcccacatc acagtaagaa ttccacacac catacaactt ggaaagaggc 160800
    tccagctgaa ggaagcccca cacttctttc aagtttttct tagtcttctc ttcttggcaa 160860
    agagtacctt ttgtttcttc taattatgta actattggtt tagtaaatat tcacccattc 160920
    agtcaccctg taagtggcag gcactgttta cagggacaca ggaaggaata aaaacttgca 160980
    ggcaccttgg agcttgcatt ctattgaaga ggtaatggaa gttgggatag cagctaaact 161040
    atgctggtat tggccaggcg cagtggctca cacctgtaat cccagcactt tggaggccaa 161100
    ggtgggcaga tcatgaagtc aggagatcga gaccatcctg gctaacatgg tgaaaccccg 161160
    tctctactaa aagtaaaaaa aaaaattagc caggtgtggt ggcgggcgcc tgtagtccca 161220
    gctacttggg aggctgaggc aggagaatgg tgtgaaccca ggaggcgaag attgcagtga 161280
    gccgagatgg caccactgca ctccagcctg ggtgacagag cgagactctg tctcagaaaa 161340
    aaaaaatatg ctggtagttt tgattcaaga tggcctttgg agcccatgat ttaggtctcg 161400
    tacccaccaa ggtctactgg aaaacatcag gctctcctgc tatagaccca tagggagagc 161460
    tgcagccgag agggggagct gaagagaagt gccccttctg tgtcctgtca gcctcatcct 161520
    tccgcaagga ccagttgctg tgccactcca ttcacttgct gcaagactgg aggtttttcc 161580
    tcaggtgttg agcacctggt ttacaagatg tcagcatctt gatgcctgag accatcaagg 161640
    caagtctctg aacagggctt accttagagt aaggcttaga agaggccgta aagtcagtct 161700
    cagctccgtg gctctgcaga gctttgggac atgtgaattc ttaaaaacaa gactattgta 161760
    cagttactat atgcatgcag tataaaatta taaccttgga aaatcctagc tagctgttga 161820
    gctaattcca taaagtaatc agctcctgag ttctgcagtg gtaataataa tcagcataat 161880
    gagtaaacac tgtgtgtgcc aggcagcgtc tcatttgatc cttgtgataa tcttgtaagt 161940
    actgattttc tcccttcttt aaacaaagtt tttttttttt ttttagagag ggtctcacta 162000
    tgttgcccag gctagtcttg aattc 162025
    <210> SEQ ID NO 37
    <211> LENGTH: 1350
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (213)...(920)
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: GenBank AJ242973
    <309> DATABASE ENTRY DATE: 1999-10-26
    <400> SEQUENCE: 37
    gcggccgcgt cgacgtgaca gccggtacgc ccgggtttgg gcaacctcga ttacgggcgg 60
    cctccaggcc cgccagcagc gccccgcgcc gcccgcccgc gcccctgccg ccccccggtt 120
    ccggccgcgg accccactct ctgccgttcc ggctgcggct ccgctgccgg tagcgccgtc 180
    ccccgggacc acccttcggc tggcgccctc cc atg ctc tcg gcc acc cgg agg 233
    Met Leu Ser Ala Thr Arg Arg
    1 5
    gct tgc cag ctc ctc ctc ctc cac agc ctc ttt ccc gtc ccg agg atg 281
    Ala Cys Gln Leu Leu Leu Leu His Ser Leu Phe Pro Val Pro Arg Met
    10 15 20
    ggc aac tcg gcc tcg aac atc gtc agc ccc cag gag gcc ttg ccg ggc 329
    Gly Asn Ser Ala Ser Asn Ile Val Ser Pro Gln Glu Ala Leu Pro Gly
    25 30 35
    cgg aag gaa cag acc cct gta gcg gcc aaa cat cat gtc aat ggc aac 377
    Arg Lys Glu Gln Thr Pro Val Ala Ala Lys His His Val Asn Gly Asn
    40 45 50 55
    aga aca gtc gaa cct ttc cca gag gga aca cag atg gct gta ttt gga 425
    Arg Thr Val Glu Pro Phe Pro Glu Gly Thr Gln Met Ala Val Phe Gly
    60 65 70
    atg gga tgt ttc tgg gga gct gaa agg aaa ttc tgg gtc ttg aaa gga 473
    Met Gly Cys Phe Trp Gly Ala Glu Arg Lys Phe Trp Val Leu Lys Gly
    75 80 85
    gtg tat tca act caa gtt ggt ttt gca gga ggc tat act tca aat cct 521
    Val Tyr Ser Thr Gln Val Gly Phe Ala Gly Gly Tyr Thr Ser Asn Pro
    90 95 100
    act tat aaa gaa gtc tgc tca gaa aaa act ggc cat gca gaa gtc gtc 569
    Thr Tyr Lys Glu Val Cys Ser Glu Lys Thr Gly His Ala Glu Val Val
    105 110 115
    cga gtg gtg tac cag cca gaa cac atg agt ttt gag gaa ctg ctc aag 617
    Arg Val Val Tyr Gln Pro Glu His Met Ser Phe Glu Glu Leu Leu Lys
    120 125 130 135
    gtc ttc tgg gag aat cac gac ccg acc caa ggt atg cgc cag ggg aac 665
    Val Phe Trp Glu Asn His Asp Pro Thr Gln Gly Met Arg Gln Gly Asn
    140 145 150
    gac cat ggc act cag tac cgc tcg gcc atc tac ccg acc tct gcc aag 713
    Asp His Gly Thr Gln Tyr Arg Ser Ala Ile Tyr Pro Thr Ser Ala Lys
    155 160 165
    caa atg gag gca gcc ctg agc tcc aaa gag aac tac caa aag gtt ctt 761
    Gln Met Glu Ala Ala Leu Ser Ser Lys Glu Asn Tyr Gln Lys Val Leu
    170 175 180
    tca gag cac ggc ttc ggc ccc atc act acc gac atc cgg gag gga cag 809
    Ser Glu His Gly Phe Gly Pro Ile Thr Thr Asp Ile Arg Glu Gly Gln
    185 190 195
    act ttc tac tat gcg gaa gac tac cac cag cag tac ctg agc aag aac 857
    Thr Phe Tyr Tyr Ala Glu Asp Tyr His Gln Gln Tyr Leu Ser Lys Asn
    200 205 210 215
    ccc aat ggc tac tgc ggc ctt ggg ggc acc ggc gtg tcc tgc cca gtg 905
    Pro Asn Gly Tyr Cys Gly Leu Gly Gly Thr Gly Val Ser Cys Pro Val
    220 225 230
    ggt att aaa aaa taa ttgctcccca catggtgggc ctttgaggtt ccagtaaaaa 960
    Gly Ile Lys Lys *
    235
    tgctttcaac aaattgggca atgcttgtgt gattcacaat cgtggcattt aaagtgcaca 1020
    aagtacaaag gaatttatac agattgggtt taccgaagta taatctatag gaggcgcgat 1080
    ggcaagttga taaaatgtga cttatctcct aataagttat ggtgggagtg gagctgtgcg 1140
    gtttcctgtg tcttctgggg tctgagtgaa gatagcaggg atgctgtgtt cacccttctt 1200
    ggtagaagct aaggtgtgag ctgggaggtt gctggacagg atgggggacc ccagaagtcc 1260
    tttatctgtg ctctctgccc gccagtgcct tacaatttgc aaacgtgtat agcctcagtg 1320
    actcattcgc tgaaatcctt cgctttacca 1350
    <210> SEQ ID NO 38
    <211> LENGTH: 235
    <212> TYPE: PRT
    <213> ORGANISM: Homo Sapien
    <400> SEQUENCE: 38
    Met Leu Ser Ala Thr Arg Arg Ala Cys Gln Leu Leu Leu Leu His Ser
    1 5 10 15
    Leu Phe Pro Val Pro Arg Met Gly Asn Ser Ala Ser Asn Ile Val Ser
    20 25 30
    Pro Gln Glu Ala Leu Pro Gly Arg Lys Glu Gln Thr Pro Val Ala Ala
    35 40 45
    Lys His His Val Asn Gly Asn Arg Thr Val Glu Pro Phe Pro Glu Gly
    50 55 60
    Thr Gln Met Ala Val Phe Gly Met Gly Cys Phe Trp Gly Ala Glu Arg
    65 70 75 80
    Lys Phe Trp Val Leu Lys Gly Val Tyr Ser Thr Gln Val Gly Phe Ala
    85 90 95
    Gly Gly Tyr Thr Ser Asn Pro Thr Tyr Lys Glu Val Cys Ser Glu Lys
    100 105 110
    Thr Gly His Ala Glu Val Val Arg Val Val Tyr Gln Pro Glu His Met
    115 120 125
    Ser Phe Glu Glu Leu Leu Lys Val Phe Trp Glu Asn His Asp Pro Thr
    130 135 140
    Gln Gly Met Arg Gln Gly Asn Asp His Gly Thr Gln Tyr Arg Ser Ala
    145 150 155 160
    Ile Tyr Pro Thr Ser Ala Lys Gln Met Glu Ala Ala Leu Ser Ser Lys
    165 170 175
    Glu Asn Tyr Gln Lys Val Leu Ser Glu His Gly Phe Gly Pro Ile Thr
    180 185 190
    Thr Asp Ile Arg Glu Gly Gln Thr Phe Tyr Tyr Ala Glu Asp Tyr His
    195 200 205
    Gln Gln Tyr Leu Ser Lys Asn Pro Asn Gly Tyr Cys Gly Leu Gly Gly
    210 215 220
    Thr Gly Val Ser Cys Pro Val Gly Ile Lys Lys
    225 230 235
    <210> SEQ ID NO 39
    <211> LENGTH: 481
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: GenBank AW195104
    <309> DATABASE ENTRY DATE: 1999-11-29
    <400> SEQUENCE: 39
    ggcattattg gactgtaggt ttttattaaa acaaacattt ctcatagctc taagcaaagc 60
    attagaattc atcaagcgga ctcacatctt ttctctgcac agagaggggc tgaaaaggga 120
    gagaaagtcc cttatgtatg tctagatttg gtaaagcgaa ggatttcagc gaatgagtca 180
    ctgaggctat acacgtttgc aaattgtaag gcactggcgg gcagagagca cagataaagg 240
    acttctgggg tcccccatcc tgtccagcaa cctcccagct cacaccttag cttctaccaa 300
    gaagggtgaa cacagcatcc ctgctatctt cactcagacc ccagaaaacc cagggaaacc 360
    cgacagctcc actcccacca taacttatta ggagataagt cacattttat caacttgcca 420
    tcgcgcctcc tatagattat acttcggtaa acccaatctg tataaattcc tttgtacttt 480
    g 481
    <210> SEQ ID NO 40
    <211> LENGTH: 390
    <212> TYPE: DNA
    <213> ORGANISM: Homo Sapien
    <300> PUBLICATION INFORMATION:
    <308> DATABASE ACCESSION NUMBER: GenBank AW874187
    <309> DATABASE ENTRY DATE: 2000-05-22
    <400> SEQUENCE: 40
    ttttttttat tggactgtag gtttttatta aaacaaacat ttctcatagc tctaagcaaa 60
    gcattagaat tcatcaagcg gactcacatc ttttctctgc acagagaggg ctgaaaaggg 120
    agagaaagcc ccttatgtat gtctagattt ggtaaagcga aggatttcag cgaatgagtc 180
    actgaggcta tacacgtttg caaattgtaa ggcactggcg ggcagagagc acagataaag 240
    gacttttggg ggtcccccat tcctgtccag caacctccca gctcacacct tagcttctac 300
    caagaagggg tgaacacagc atccctgcta tcttcactca gacccccaga agacacagga 360
    aaccgcacag ctccactccc accataactt 390
    <210> SEQ ID NO 41
    <211> LENGTH: 43
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 41
    agcggataac aatttcacac agggagctag cttggaagat tgc 43
    <210> SEQ ID NO 42
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 42
    gtccaatata tgcaaacagt tg 22
    <210> SEQ ID NO 43
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 43
    agcggataac aatttcacac agg 23
    <210> SEQ ID NO 44
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 44
    actgagcctg ctgcataa 18
    <210> SEQ ID NO 45
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 45
    tctcaatcat gtgcattgag g 21
    <210> SEQ ID NO 46
    <211> LENGTH: 43
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 46
    agcggataac aatttcacac agggatcaca cagccatcag cag 43
    <210> SEQ ID NO 47
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: oligonucleotide primer
    <400> SEQUENCE: 47
    agcggataac aatttcacac agg 23
    <210> SEQ ID NO 48
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Oligonucleotide primer
    <400> SEQUENCE: 48
    ctggcgccac gtggtcaa 18
    <210> SEQ ID NO 49
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 49
    tttctctgca cagagagggc 20
    <210> SEQ ID NO 50
    <211> LENGTH: 44
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 50
    agcggataac aatttcacac agggctgaaa tccttcgctt tacc 44
    <210> SEQ ID NO 51
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 51
    agcggataac aatttcacac agg 23
    <210> SEQ ID NO 52
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 52
    ctgaaaaggg agagaaag 18
    <210> SEQ ID NO 53
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 53
    tcccaaagtg ctggaattac 20
    <210> SEQ ID NO 54
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 54
    gtccaatata tgcaaacagt tg 22
    <210> SEQ ID NO 55
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Primer
    <400> SEQUENCE: 55
    cccacagcag ttaatccttc 20
    <210> SEQ ID NO 56
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 56
    gcgctcctgt cggtgcca 18
    <210> SEQ ID NO 57
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 57
    gcctgactgg tggggccc 18
    <210> SEQ ID NO 58
    <211> LENGTH: 15
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 58
    catgcatgca cggtc 15
    <210> SEQ ID NO 59
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 59
    cagagagtac ccctcgaccg tgcatgcatg 30
    <210> SEQ ID NO 60
    <211> LENGTH: 15
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 60
    catgcatgca cggtt 15
    <210> SEQ ID NO 61
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 61
    gtacgtacgt gccaactccc catgagagac 30
    <210> SEQ ID NO 62
    <211> LENGTH: 14
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 62
    catgcatgca cggt 14
    <210> SEQ ID NO 63
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 63
    gcctgactgg tggggccc 18
    <210> SEQ ID NO 64
    <211> LENGTH: 26
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 64
    gtgctgcagg tgtaaacttg taccag 26
    <210> SEQ ID NO 65
    <211> LENGTH: 28
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 65
    cacggatccg gtagcagcgg tagagttg 28
    <210> SEQ ID NO 66
    <211> LENGTH: 19
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 66
    actgggcatg tggagacag 19
    <210> SEQ ID NO 67
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 67
    gcactttctt gccatgag 18
    <210> SEQ ID NO 68
    <211> LENGTH: 14
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 68
    tcagtcacga cgtt 14
    <210> SEQ ID NO 69
    <211> LENGTH: 14
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 69
    cggataacaa tttc 14
    <210> SEQ ID NO 70
    <211> LENGTH: 37
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 70
    caatttcatc gctggatgca atctgggcta tgagatc 37
    <210> SEQ ID NO 71
    <211> LENGTH: 37
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 71
    caatttcaca cagcggatgc ttcttttggc tctgact 37
    <210> SEQ ID NO 72
    <211> LENGTH: 40
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 72
    tcagtcacga cgttggatgc caataaaagt gactctcagc 40
    <210> SEQ ID NO 73
    <211> LENGTH: 37
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 73
    cggataacaa tttcggatgc actgggagca ttgaggc 37
    <210> SEQ ID NO 74
    <211> LENGTH: 38
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 74
    tcagtcacga cgttggatga gcagatccct ggacaggc 38
    <210> SEQ ID NO 75
    <211> LENGTH: 38
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 75
    cggataacaa tttcggatgg acaaaatacc tgtattcc 38
    <210> SEQ ID NO 76
    <211> LENGTH: 36
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 76
    tcagtcacga cgttggatgc agagcagctc cgagtc 36
    <210> SEQ ID NO 77
    <211> LENGTH: 32
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 77
    cagcggtgat cattggatgc aggaagctct gg 32
    <210> SEQ ID NO 78
    <211> LENGTH: 38
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 78
    tcagtcacga cgttggatgc ccacatgcca cccactac 38
    <210> SEQ ID NO 79
    <211> LENGTH: 35
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 79
    cggataacaa tttcggatgc ccgtcaggta ccacg 35
    <210> SEQ ID NO 80
    <211> LENGTH: 37
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 80
    tcagtcacga cgttggatgc ccacagtgga gcttcag 37
    <210> SEQ ID NO 81
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 81
    gctcatacct tgcaggatga cg 22
    <210> SEQ ID NO 82
    <211> LENGTH: 36
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 82
    tcagtcacga cgttggatga ccagctgttc gtgttc 36
    <210> SEQ ID NO 83
    <211> LENGTH: 34
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 83
    tacatggagt tcggggatgc acacggcgac tctc 34
    <210> SEQ ID NO 84
    <211> LENGTH: 40
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 84
    tcagtcacga cgttggatgg ggaagagcag agatatacgt 40
    <210> SEQ ID NO 85
    <211> LENGTH: 29
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 85
    gaggggctga tccaggatgg gtgctccac 29
    <210> SEQ ID NO 86
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 86
    tgaagcactt gaaggatgag ggtgtctgcg 30
    <210> SEQ ID NO 87
    <211> LENGTH: 38
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 87
    cggataacaa tttcggatgc tgcgtgatga tgaaatcg 38
    <210> SEQ ID NO 88
    <211> LENGTH: 26
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 88
    gatgaagctc ccaggatgcc agaggc 26
    <210> SEQ ID NO 89
    <211> LENGTH: 27
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 89
    gccgccggtg taggatgctg ctggtgc 27
    <210> SEQ ID NO 90
    <211> LENGTH: 31
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide Template
    <400> SEQUENCE: 90
    cgcagggttt cctcgtcgca ctgggcatgt g 31
    <210> SEQ ID NO 91
    <211> LENGTH: 43
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Biotinylatd primer
    <400> SEQUENCE: 91
    tgcttatccc tgtagctacc ctgtcttggc cttgcagatc caa 43
    <210> SEQ ID NO 92
    <211> LENGTH: 42
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 92
    agcggataac aatttcacac aggccatcac accgcggtac tg 42
    <210> SEQ ID NO 93
    <211> LENGTH: 44
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 93
    cccagtcacg acgttgtaaa acgtcttggc cttgcagatc caag 44
    <210> SEQ ID NO 94
    <211> LENGTH: 42
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 94
    agcggataac aatttcacac aggccatcac accgcggtac tg 42
    <210> SEQ ID NO 95
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 95
    ctccagctgg gcaggagtgc 20
    <210> SEQ ID NO 96
    <211> LENGTH: 17
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Oligonucleotide primer
    <400> SEQUENCE: 96
    cacttcagtc gctccct 17
    <210> SEQ ID NO 97
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Biotinylated primer
    <400> SEQUENCE: 97
    cccagtcacg acgttgtaaa acg 23
    <210> SEQ ID NO 98
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 98
    cctttgagaa agggctctgc ttgagttgta gaaagaaccg ctgcaacaat ctgggctatg 60
    agatcaataa agtcagagcc aaaagaagca gcaaaatgta 100
    <210> SEQ ID NO 99
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 99
    cctttgagaa agggctctgc ttgagttgta gaaagaaccg ctgcaacaat ctgggctatg 60
    agatcagtaa agtcagagcc aaaagaagca gcaaaatgta 100
    <210> SEQ ID NO 100
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 100
    gaattatttt tgtgtttcta aaactatggt tcccaataaa agtgactctc agcgagcctc 60
    aatgctccca gtgctattca tgggcagctc tctgggctca 100
    <210> SEQ ID NO 101
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 101
    gaattatttt tgtgtttcta aaactatggt tcccaataaa agtgactctc agcaagcctc 60
    aatgctccca gtgctattca tgggcagctc tctgggctca 100
    <210> SEQ ID NO 102
    <211> LENGTH: 84
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 102
    taataggact acttctaatc tgtaagagca gatccctgga caggcgagga atacaggtat 60
    tttgtccttg aagtaacctt tcag 84
    <210> SEQ ID NO 103
    <211> LENGTH: 84
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 103
    taataggact acttctaatc tgtaagagca gatccctgga caggcaagga atacaggtat 60
    tttgtccttg aagtaacctt tcag 84
    <210> SEQ ID NO 104
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 104
    ctcaccatgg gcatttgatt gcagagcagc tccgagtccg tccagagctt cctgcagtca 60
    atgatcaccg ctgtgggcat ccctgaggtc atgtctcgta 100
    <210> SEQ ID NO 105
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 105
    ctcaccatgg gcatttgatt gcagagcagc tccgagtcca tccagagctt cctgcagtca 60
    atgatcaccg ctgtgggcat ccctgaggtc atgtctcgta 100
    <210> SEQ ID NO 106
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 106
    agcaaggact cctgcaaggg ggacagtgga ggcccacatg ccacccacta ccagggcacg 60
    tggtacctga cgggcatcgt cagctggggc cagggctgcg 100
    <210> SEQ ID NO 107
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 107
    agcaaggact cctgcaaggg ggacagtgga ggcccacatg ccacccacta ccggggcacg 60
    tggtacctga cgggcatcgt cagctggggc cagggctgcg 100
    <210> SEQ ID NO 108
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Hom sapien
    <400> SEQUENCE: 108
    caataactct aatgcagcgg aagatgacct gcccacagtg gagcttcagg gcgtggtgcc 60
    ccggggcgtc aacctgcaag gtatgagcat accccccttc 100
    <210> SEQ ID NO 109
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 109
    caataactct aatgcagcgg aagatgacct gcccacagtg gagcttcagg gcttggtgcc 60
    ccggggcgtc aacctgcaag gtatgagcat accccccttc 100
    <210> SEQ ID NO 110
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 110
    ttgaagcttt gggctacgtg gatgaccagc tgttcgtgtt ctatgatcat gagagtcgcc 60
    gtgtggagcc ccgaactcca tgggtttcca gtagaatttc 100
    <210> SEQ ID NO 111
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 111
    ttgaagcttt gggctacgtg gatgaccagc tgttcgtgtt ctatgatgat gagagtcgcc 60
    gtgtggagcc ccgaactcca tgggtttcca gtagaatttc 100
    <210> SEQ ID NO 112
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 112
    ggataacctt ggctgtaccc cctggggaag agcagagata tacgtgccag gtggagcacc 60
    caggcctgga tcagcccctc attgtgatct gggagccctc 100
    <210> SEQ ID NO 113
    <211> LENGTH: 100
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 113
    ggataacctt ggctgtaccc cctggggaag agcagagata tacgtaccag gtggagcacc 60
    caggcctgga tcagcccctc attgtgatct gggagccctc 100
    <210> SEQ ID NO 114
    <211> LENGTH: 80
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 114
    tgaagcactt gaaggagaag gtgtctgcgg gagccgattt catcatcacg cagcttttct 60
    ttgaggctga cacattcttc 80
    <210> SEQ ID NO 115
    <211> LENGTH: 80
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 115
    tgaagcactt gaaggagaag gtgtctgcgg gagtcgattt catcatcacg cagcttttct 60
    ttgaggctga cacattcttc 80
    <210> SEQ ID NO 116
    <211> LENGTH: 80
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 116
    tccagatgaa gctcccagaa tgccagaggc tgctccccgc gtggcccctg caccagcagc 60
    tcctacaccg gcggcccctg 80
    <210> SEQ ID NO 117
    <211> LENGTH: 80
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapien
    <400> SEQUENCE: 117
    tccagatgaa gctcccagaa tgccagaggc tgctcccccc gtggcccctg caccagcagc 60
    tcctacaccg gcggcccctg 80
    <210> SEQ ID NO 118
    <211> LENGTH: 48
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Hair pin structure
    <400> SEQUENCE: 118
    cagagagtac ccctcaaccg tgcatgcatg aaacatgcat gcacggtt 48

Claims (3)

What is claimed is:
1. A high throughput method of determining frequencies of genetic variations, comprising:
selecting a healthy target population and a genetic variation to be assessed;
pooling a plurality of samples of biopolymers obtained from members of the population;
determining or detecting the biopolymer that comprises the variation by mass spectrometry;
obtaining a mass spectrum or a digital representation thereof; and
determining the frequency of the variation in the population.
2. The method of claim 1, wherein:
the variation is selected from the group consisting of an allelic variation, a post-translational modification, a nucleic modification, a label, a mass modification of a nucleic acid and methylation; and/or
the biopolymer is a nucleic acid, a protein, a polysaccharide, a lipid, a small organic metabolite or intermediate, wherein the concentration of biopolymer of interest is the same in each of the samples; and/or
the frequency is determined by assessing the method comprising determining the area under the peak in the mass spectrum or digital representation thereof corresponding to the mass of the biopolymer comprising the genomic variation.
3. The method of claim 2, wherein the method for determining the frequency is effected by determining the ratio of the signal or the digital representation thereof to the total area of the entire mass spectrum, which is corrected for background.
US10/272,756 1999-10-13 2002-10-15 Methods for generating databases and databases for identifying polymorphic genetic markers Abandoned US20030190644A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/272,756 US20030190644A1 (en) 1999-10-13 2002-10-15 Methods for generating databases and databases for identifying polymorphic genetic markers

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US15917699P 1999-10-13 1999-10-13
US21725100P 2000-07-10 2000-07-10
US21765800P 2000-07-10 2000-07-10
US09/663,968 US7917301B1 (en) 2000-09-19 2000-09-19 Method and device for identifying a biological sample
US68748300A 2000-10-13 2000-10-13
US10/272,756 US20030190644A1 (en) 1999-10-13 2002-10-15 Methods for generating databases and databases for identifying polymorphic genetic markers

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
US09/663,968 Continuation-In-Part US7917301B1 (en) 1999-10-13 2000-09-19 Method and device for identifying a biological sample
US68748300A Division 1999-10-13 2000-10-13

Publications (1)

Publication Number Publication Date
US20030190644A1 true US20030190644A1 (en) 2003-10-09

Family

ID=46281349

Family Applications (6)

Application Number Title Priority Date Filing Date
US10/272,665 Expired - Lifetime US7332275B2 (en) 1999-10-13 2002-10-15 Methods for detecting methylated nucleotides
US10/273,321 Expired - Fee Related US7668658B2 (en) 1999-10-13 2002-10-15 Methods for generating databases and databases for identifying polymorphic genetic markers
US10/272,756 Abandoned US20030190644A1 (en) 1999-10-13 2002-10-15 Methods for generating databases and databases for identifying polymorphic genetic markers
US12/643,933 Expired - Fee Related US8229677B2 (en) 1999-10-13 2009-12-21 Methods for generating databases and databases for identifying polymorphic genetic markers
US13/536,807 Expired - Fee Related US8818735B2 (en) 1999-10-13 2012-06-28 Methods for generating databases and databases for identifying polymorphic genetic markers
US14/274,476 Abandoned US20150005194A1 (en) 1999-10-13 2014-05-09 Methods for generating databases and databases for identifying polymorphic genetic markers

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US10/272,665 Expired - Lifetime US7332275B2 (en) 1999-10-13 2002-10-15 Methods for detecting methylated nucleotides
US10/273,321 Expired - Fee Related US7668658B2 (en) 1999-10-13 2002-10-15 Methods for generating databases and databases for identifying polymorphic genetic markers

Family Applications After (3)

Application Number Title Priority Date Filing Date
US12/643,933 Expired - Fee Related US8229677B2 (en) 1999-10-13 2009-12-21 Methods for generating databases and databases for identifying polymorphic genetic markers
US13/536,807 Expired - Fee Related US8818735B2 (en) 1999-10-13 2012-06-28 Methods for generating databases and databases for identifying polymorphic genetic markers
US14/274,476 Abandoned US20150005194A1 (en) 1999-10-13 2014-05-09 Methods for generating databases and databases for identifying polymorphic genetic markers

Country Status (1)

Country Link
US (6) US7332275B2 (en)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030096426A1 (en) * 1997-01-23 2003-05-22 Daniel P. Little Systems and methods for preparing and analyzing low volume analyte array elements
WO2005020788A2 (en) * 2003-08-01 2005-03-10 The General Hospital Corporation Cognition analysis
WO2005098050A2 (en) 2004-03-26 2005-10-20 Sequenom, Inc. Base specific cleavage of methylation-specific amplification products in combination with mass analysis
US20050260603A1 (en) * 2002-12-31 2005-11-24 Mmi Genomics, Inc. Compositions for inferring bovine traits
US7668658B2 (en) 1999-10-13 2010-02-23 Sequenom, Inc. Methods for generating databases and databases for identifying polymorphic genetic markers
US20100162423A1 (en) * 2003-10-24 2010-06-24 Metamorphix, Inc. Methods and Systems for Inferring Traits to Breed and Manage Non-Beef Livestock
US7759065B2 (en) 1995-03-17 2010-07-20 Sequenom, Inc. Mass spectrometric methods for detecting mutations in a target nucleic acid
US7820378B2 (en) 2002-11-27 2010-10-26 Sequenom, Inc. Fragmentation-based methods and systems for sequence variation detection and discovery
US8999266B2 (en) 2000-10-30 2015-04-07 Agena Bioscience, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate
US9068953B2 (en) 2007-09-17 2015-06-30 Agena Bioscience, Inc. Integrated robotic sample transfer device
US9394565B2 (en) 2003-09-05 2016-07-19 Agena Bioscience, Inc. Allele-specific sequence variation analysis
US20210017592A1 (en) * 2017-07-07 2021-01-21 Massachusetts Institute Of Technology Systems and methods for genetic identification and analysis

Families Citing this family (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6994969B1 (en) * 1999-04-30 2006-02-07 Methexis Genomics, N.V. Diagnostic sequencing by a combination of specific cleavage and mass spectrometry
EP1268857A2 (en) * 2000-04-06 2003-01-02 Epigenomics AG Diagnosis of diseases associated with gene regulation
US20060286577A1 (en) * 2005-06-17 2006-12-21 Xiyu Jia Methods for detection of methylated DNA
US20070245184A1 (en) * 2006-03-06 2007-10-18 Applera Corporation Method and system for generating validation workflow
EP2016188B1 (en) * 2006-04-24 2015-10-21 Xiyu Jia Methods for detection of methylated dna
JP5227556B2 (en) * 2007-09-06 2013-07-03 株式会社日立製作所 Analysis equipment
WO2009114543A2 (en) 2008-03-11 2009-09-17 Sequenom, Inc. Nucleic acid-based tests for prenatal gender determination
US8831293B2 (en) * 2008-04-21 2014-09-09 Mts Investments Inc. System and method for statistical mapping between genetic information and facial image data
EP4047367A1 (en) 2008-07-18 2022-08-24 Bio-Rad Laboratories, Inc. Method for detecting target analytes with droplet libraries
US8962247B2 (en) 2008-09-16 2015-02-24 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses
US8476013B2 (en) 2008-09-16 2013-07-02 Sequenom, Inc. Processes and compositions for methylation-based acid enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
WO2010065139A1 (en) * 2008-12-05 2010-06-10 23Andme, Inc. Gamete donor selection based on genetic calculations
EP2394175B1 (en) * 2009-02-09 2016-02-03 caprotec bioanalytics GmbH Devices, systems and methods for separating magnetic particles
JP2013511991A (en) * 2009-11-25 2013-04-11 クアンタライフ, インコーポレイテッド Methods and compositions for detecting genetic material
DK2516680T3 (en) 2009-12-22 2016-05-02 Sequenom Inc Method and kits to identify aneuploidy
US8877455B2 (en) * 2010-03-24 2014-11-04 Glendon John Parker Methods for conducting genetic analysis using protein polymorphisms
CA2826748C (en) 2011-02-09 2020-08-04 Bio-Rad Laboratories, Inc. Method of detecting variations in copy number of a target nucleic acid
EP3736281A1 (en) 2011-02-18 2020-11-11 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US8455221B2 (en) 2011-04-29 2013-06-04 Sequenom, Inc. Quantification of a minority nucleic acid species
US8658430B2 (en) 2011-07-20 2014-02-25 Raindance Technologies, Inc. Manipulating droplet size
US8990250B1 (en) 2011-10-11 2015-03-24 23Andme, Inc. Cohort selection with privacy protection
CN103160937B (en) * 2011-12-15 2015-02-18 深圳华大基因科技服务有限公司 Method for conducting enrichment library construction and SNP analysis on gene of complex genome of higher plant
EP4155401A1 (en) 2012-03-02 2023-03-29 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US9920361B2 (en) 2012-05-21 2018-03-20 Sequenom, Inc. Methods and compositions for analyzing nucleic acid
AU2013290102B2 (en) 2012-07-13 2018-11-15 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
EP3597774A1 (en) 2013-03-13 2020-01-22 Sequenom, Inc. Primers for dna methylation analysis
WO2014190231A1 (en) 2013-05-23 2014-11-27 Iphenotype Llc Methods and systems for assisting persons, product providers and/or service providers
CN105555972B (en) 2013-07-25 2020-07-31 伯乐生命医学产品有限公司 Genetic assay
EP3736344A1 (en) 2014-03-13 2020-11-11 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
AU2015236054B2 (en) 2014-03-25 2019-11-21 Five3 Genomics, Llc Systems and methods for RNA analysis in functional confirmation of cancer mutations
US10395759B2 (en) 2015-05-18 2019-08-27 Regeneron Pharmaceuticals, Inc. Methods and systems for copy number variant detection
US10747899B2 (en) * 2015-10-07 2020-08-18 The Board Of Trustees Of The Leland Stanford Junior University Techniques for determining whether an individual is included in ensemble genomic data
JP7141029B2 (en) * 2017-07-12 2022-09-22 シスメックス株式会社 How to build a database
CN111325121B (en) * 2020-02-10 2024-02-20 浙江迪谱诊断技术有限公司 Nucleic acid mass spectrum numerical processing method
DE102020211219A1 (en) 2020-09-08 2022-03-10 Robert Bosch Gesellschaft mit beschränkter Haftung Method and controller for determining a number of samples for bulk analysis using an analyzer for analyzing samples of biological material
US11379578B1 (en) 2020-10-16 2022-07-05 Trend Micro Incorporated Detecting malware by pooled analysis of sample files in a sandbox
WO2023049490A1 (en) * 2021-09-27 2023-03-30 Purdue Reserach Foundation Label-free food analysis and molecular detection

Citations (56)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9394A (en) * 1852-11-09 Brick-machnsfe
US42112A (en) * 1864-03-29 Improvement in grain-drills
US155587A (en) * 1874-10-06 Improvement in billiard-cushions
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4826360A (en) * 1986-03-10 1989-05-02 Shimizu Construction Co., Ltd. Transfer system in a clean room
US4851018A (en) * 1986-11-28 1989-07-25 Commissariat A L'energie Atomique Installation for the storage and transfer of objects in a very clean atmosphere
US5118937A (en) * 1989-08-22 1992-06-02 Finnigan Mat Gmbh Process and device for the laser desorption of an analyte molecular ions, especially of biomolecules
US5436150A (en) * 1992-04-03 1995-07-25 The Johns Hopkins University Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease
US5440119A (en) * 1992-06-02 1995-08-08 Labowsky; Michael J. Method for eliminating noise and artifact peaks in the deconvolution of multiply charged mass spectra
US5453613A (en) * 1994-10-21 1995-09-26 Hewlett Packard Company Mass spectra interpretation system including spectra extraction
US5498545A (en) * 1994-07-21 1996-03-12 Vestal; Marvin L. Mass spectrometer system and method for matrix-assisted laser desorption measurements
US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization
US5506137A (en) * 1992-07-23 1996-04-09 Stratagene Purified thermostable Pyrococcus furiosus DNA ligase
US5536649A (en) * 1993-05-11 1996-07-16 Becton, Dickinson And Company Decontamination of nucleic acid amplification reactions using uracil-N-glycosylase (UDG)
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5604098A (en) * 1993-03-24 1997-02-18 Molecular Biology Resources, Inc. Methods and materials for restriction endonuclease applications
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
US5622824A (en) * 1993-03-19 1997-04-22 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5686656A (en) * 1996-02-27 1997-11-11 Aviv Amirav Method and device for the introduction of a sample into a gas chromatograph
US5714330A (en) * 1994-04-04 1998-02-03 Lynx Therapeutics, Inc. DNA sequencing by stepwise ligation and cleavage
US5777324A (en) * 1996-09-19 1998-07-07 Sequenom, Inc. Method and apparatus for maldi analysis
US5786146A (en) * 1996-06-03 1998-07-28 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US5795714A (en) * 1992-11-06 1998-08-18 Trustees Of Boston University Method for replicating an array of nucleic acid probes
US5837832A (en) * 1993-06-25 1998-11-17 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips
US5843669A (en) * 1996-01-24 1998-12-01 Third Wave Technologies, Inc. Cleavage of nucleic acid acid using thermostable methoanococcus jannaschii FEN-1 endonucleases
US5853979A (en) * 1995-06-30 1998-12-29 Visible Genetics Inc. Method and system for DNA sequence determination and mutation detection with reference to a standard
US5858705A (en) * 1995-06-05 1999-01-12 Human Genome Sciences, Inc. Polynucleotides encoding human DNA ligase III and methods of using these polynucleotides
US5871911A (en) * 1992-12-07 1999-02-16 Wisconsin Alumni Research Foundation Method of site-specific nucleic acid cleavage
US5874283A (en) * 1995-05-30 1999-02-23 John Joseph Harrington Mammalian flap-specific endonuclease
US5885841A (en) * 1996-09-11 1999-03-23 Eli Lilly And Company System and methods for qualitatively and quantitatively comparing complex admixtures using single ion chromatograms derived from spectroscopic analysis of such admixtures
US5888795A (en) * 1997-09-09 1999-03-30 Becton, Dickinson And Company Thermostable uracil DNA glycosylase and methods of use
US5900481A (en) * 1996-11-06 1999-05-04 Sequenom, Inc. Bead linkers for immobilizing nucleic acids to solid supports
US5928906A (en) * 1996-05-09 1999-07-27 Sequenom, Inc. Process for direct sequencing during template amplification
US5928870A (en) * 1997-06-16 1999-07-27 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US5952176A (en) * 1995-07-11 1999-09-14 Forfas (Trading As Bioresearch Ireland) Glycosylase mediated detection of nucleotide sequences at candidate loci
US5976806A (en) * 1997-06-25 1999-11-02 Pioneer Hi-Bred International, Inc. DNA ligase assay
US5975492A (en) * 1997-07-14 1999-11-02 Brenes; Arthur Bellows driver slot valve
US6017704A (en) * 1996-06-03 2000-01-25 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US6022688A (en) * 1996-05-13 2000-02-08 Sequenom, Inc. Method for dissociating biotin complexes
US6024925A (en) * 1997-01-23 2000-02-15 Sequenom, Inc. Systems and methods for preparing low volume analyte array elements
US6054276A (en) * 1998-02-23 2000-04-25 Macevicz; Stephen C. DNA restriction site mapping
US6059724A (en) * 1997-02-14 2000-05-09 Biosignal, Inc. System for predicting future health
US6074823A (en) * 1993-03-19 2000-06-13 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6090606A (en) * 1996-01-24 2000-07-18 Third Wave Technologies, Inc. Cleavage agents
US6099553A (en) * 1998-05-21 2000-08-08 Applied Medical Resources Corporation Suture clinch
US6133436A (en) * 1996-11-06 2000-10-17 Sequenom, Inc. Beads bound to a solid support and to nucleic acids
US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6146854A (en) * 1995-08-31 2000-11-14 Sequenom, Inc. Filtration processes, kits and devices for isolating plasmids
US6188064B1 (en) * 1998-01-29 2001-02-13 Bruker Daltonik Gmbh Mass spectrometry method for accurate mass determination of unknown ions
US6207370B1 (en) * 1997-09-02 2001-03-27 Sequenom, Inc. Diagnostics based on mass spectrometric detection of translated target polypeptides
US6268131B1 (en) * 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids
US6270835B1 (en) * 1999-10-07 2001-08-07 Microcoating Technologies, Inc. Formation of this film capacitors
US6428955B1 (en) * 1995-03-17 2002-08-06 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids
US6566055B1 (en) * 1996-09-19 2003-05-20 Sequenom, Inc. Methods of preparing nucleic acids for mass spectrometric analysis

Family Cites Families (201)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2031216A1 (en) 1969-06-19 1971-01-14 Citizen Watch Co Ltd , Tokio Day and date setting device for clocks with calendar
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US3940475A (en) 1970-06-11 1976-02-24 Biological Developments, Inc. Radioimmune method of assaying quantitatively for a hapten
NL154598B (en) 1970-11-10 1977-09-15 Organon Nv PROCEDURE FOR DETERMINING AND DETERMINING LOW MOLECULAR COMPOUNDS AND PROTEINS THAT CAN SPECIFICALLY BIND THESE COMPOUNDS AND TEST PACKAGING.
US3817837A (en) 1971-05-14 1974-06-18 Syva Corp Enzyme amplification assay
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US3939350A (en) 1974-04-29 1976-02-17 Board Of Trustees Of The Leland Stanford Junior University Fluorescent immunoassay employing total reflection for activation
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
DE2548891C3 (en) 1975-10-31 1983-04-28 Finnigan MAT GmbH, 2800 Bremen Sample changer for mass spectrometers
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4277437A (en) 1978-04-05 1981-07-07 Syva Company Kit for carrying out chemically induced fluorescence immunoassay
JPS6023084B2 (en) 1979-07-11 1985-06-05 味の素株式会社 blood substitute
US4366241A (en) 1980-08-07 1982-12-28 Syva Company Concentrating zone method in heterogeneous immunoassays
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4722848A (en) 1982-12-08 1988-02-02 Health Research, Incorporated Method for immunizing animals with synthetically modified vaccinia virus
US4562639A (en) 1982-03-23 1986-01-07 Texas Instruments Incorporated Process for making avalanche fuse element with isolated emitter
US4511503A (en) 1982-12-22 1985-04-16 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins
US4568649A (en) 1983-02-22 1986-02-04 Immunex Corporation Immediate ligand detection assay
GB8311018D0 (en) 1983-04-22 1983-05-25 Amersham Int Plc Detecting mutations in dna
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
FR2567892B1 (en) 1984-07-19 1989-02-17 Centre Nat Rech Scient NOVEL OLIGONUCLEOTIDES, THEIR PREPARATION PROCESS AND THEIR APPLICATIONS AS MEDIATORS IN DEVELOPING THE EFFECTS OF INTERFERONS
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5173418A (en) 1985-05-10 1992-12-22 Benzon Pharma, A/S Production in Escherichia coli of extracellular Serratia spp. hydrolases
DE3675588D1 (en) 1985-06-19 1990-12-20 Ajinomoto Kk HAEMOGLOBIN TIED TO A POLY (ALKENYLENE OXIDE).
US5079342A (en) 1986-01-22 1992-01-07 Institut Pasteur Cloned DNA sequences related to the entire genomic RNA of human immunodeficiency virus II (HIV-2), polypeptides encoded by these DNA sequences and use of these DNA clones and polypeptides in diagnostic kits
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US4726360A (en) * 1986-07-17 1988-02-23 Medical Engineering Corporation Penile prosthesis
EP0260032B1 (en) 1986-09-08 1994-01-26 Ajinomoto Co., Inc. Compounds for the cleavage at a specific position of RNA, oligomers employed for the formation of said compounds, and starting materials for the synthesis of said oligomers
US4998617A (en) 1986-09-15 1991-03-12 Laura Lupton Inc Facial cosmetic liquid make up kit
US4987071A (en) 1986-12-03 1991-01-22 University Patents, Inc. RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods
US5202231A (en) 1987-04-01 1993-04-13 Drmanac Radoje T Method of sequencing of genomes by hybridization of oligonucleotide probes
US6270961B1 (en) 1987-04-01 2001-08-07 Hyseq, Inc. Methods and apparatus for DNA sequencing and DNA identification
US5525464A (en) 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes
US4837726A (en) 1987-06-19 1989-06-06 Applied Biosystems, Inc. Quantitation of chromatographic information
US5024939A (en) 1987-07-09 1991-06-18 Genentech, Inc. Transient expression system for producing recombinant protein
US4802102A (en) 1987-07-15 1989-01-31 Hewlett-Packard Company Baseline correction for chromatography
ATE151467T1 (en) 1987-11-30 1997-04-15 Univ Iowa Res Found DNA MOLECULES STABILIZED BY MODIFICATIONS TO THE 3'-TERMINAL PHOSPHODIESTER BOND, THEIR USE AS NUCLEIC ACID PROBE AND AS THERAPEUTIC AGENTS FOR INHIBITING THE EXPRESSION OF SPECIFIC TARGET GENES
US5403711A (en) 1987-11-30 1995-04-04 University Of Iowa Research Foundation Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved
US4988617A (en) 1988-03-25 1991-01-29 California Institute Of Technology Method of detecting a nucleotide change in nucleic acids
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
GB8816982D0 (en) 1988-07-16 1988-08-17 Probus Biomedical Ltd Bio-fluid assay apparatus
US5025939A (en) 1988-09-16 1991-06-25 Bunn-O-Matic Corporation Coffee decanter with integral handle
US5856092A (en) 1989-02-13 1999-01-05 Geneco Pty Ltd Detection of a nucleic acid sequence or a change therein
US5082767A (en) 1989-02-27 1992-01-21 Hatfield G Wesley Codon pair utilization
EP0395481A3 (en) 1989-04-25 1991-03-20 Spectra-Physics, Inc. Method and apparatus for estimation of parameters describing chromatographic peaks
US5256775A (en) 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
US5925525A (en) 1989-06-07 1999-07-20 Affymetrix, Inc. Method of identifying nucleotide differences
US5547839A (en) 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
FR2650840B1 (en) 1989-08-11 1991-11-29 Bertin & Cie RAPID DETECTION AND / OR IDENTIFICATION OF A SINGLE BASED ON A NUCLEIC ACID SEQUENCE, AND ITS APPLICATIONS
US5591722A (en) 1989-09-15 1997-01-07 Southern Research Institute 2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
DK0942000T3 (en) 1989-10-24 2004-11-01 Isis Pharmaceuticals Inc 2'-modified oligonucleotides
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5128448A (en) 1990-01-10 1992-07-07 Hoffman-La Roche Inc. CCK analogs with appetite regulating activity
US5646265A (en) 1990-01-11 1997-07-08 Isis Pharmceuticals, Inc. Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5670633A (en) 1990-01-11 1997-09-23 Isis Pharmaceuticals, Inc. Sugar modified oligonucleotides that detect and modulate gene expression
US5623065A (en) 1990-08-13 1997-04-22 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
WO1991010674A1 (en) 1990-01-12 1991-07-25 Scripps Clinic And Research Foundation Nucleic acid enzymes for cleaving dna
US5283173A (en) 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
NZ236819A (en) 1990-02-03 1993-07-27 Max Planck Gesellschaft Enzymatic cleavage of fusion proteins; fusion proteins; recombinant dna and pharmaceutical compositions
US5220007A (en) 1990-02-15 1993-06-15 The Worcester Foundation For Experimental Biology Method of site-specific alteration of RNA and production of encoded polypeptides
US5149797A (en) 1990-02-15 1992-09-22 The Worcester Foundation For Experimental Biology Method of site-specific alteration of rna and production of encoded polypeptides
US6013431A (en) 1990-02-16 2000-01-11 Molecular Tool, Inc. Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators
IT1239733B (en) 1990-02-23 1993-11-15 Eniricerche Spa NEUTRAL THERMO-STABLE MUTANTS AND MEANS AND METHODS FOR THEIR PREPARATION
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
GB9009980D0 (en) 1990-05-03 1990-06-27 Amersham Int Plc Phosphoramidite derivatives,their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
CA2079229C (en) 1990-05-09 1999-09-14 Alexander J. Varshavsky Ubiquitin-specific protease
ES2116977T3 (en) 1990-05-11 1998-08-01 Microprobe Corp SOLID SUPPORTS FOR NUCLEIC ACID HYBRIDIZATION TESTS AND METHODS TO IMMOBILIZE OLIGONUCLEOTIDES IN A COVALENT WAY.
JP3208486B2 (en) 1990-07-02 2001-09-10 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Analyte detection using fluorescence energy transfer
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
IL113519A (en) 1990-08-03 1997-11-20 Sterling Winthrop Inc Oligonucleoside sequences of from about 6 to about 200 bases having a three atom internucleoside linkage, their preparation and pharmaceutical compositions for inhibiting gene expression containing said oligonucleosides
SE9002579D0 (en) 1990-08-07 1990-08-07 Pharmacia Ab METHOD AND APPARATUS FOR CARRYING OUT BIOCHEMICAL REACTIONS
US5264563A (en) 1990-08-24 1993-11-23 Ixsys Inc. Process for synthesizing oligonucleotides with random codons
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
AU662298B2 (en) 1990-09-20 1995-08-31 Gilead Sciences, Inc. Modified internucleoside linkages
US6004744A (en) 1991-03-05 1999-12-21 Molecular Tool, Inc. Method for determining nucleotide identity through extension of immobilized primer
US5578443A (en) 1991-03-06 1996-11-26 Regents Of The University Of Minnesota DNA sequence-based HLA typing method
CA2066556A1 (en) 1991-04-26 1992-10-27 Toyoji Sawayanagi Alkaline protease, method for producing the same, use thereof and microorganism producing the same
JPH0534650A (en) 1991-05-10 1993-02-12 Fujitsu Ltd Branch interference type optical modulator provided with monitor
US5175430A (en) 1991-05-17 1992-12-29 Meridian Instruments, Inc. Time-compressed chromatography in mass spectrometry
DE4214112A1 (en) 1991-08-02 1993-02-04 Europ Lab Molekularbiolog NEW METHOD FOR SEQUENCING NUCLEIC ACIDS
US5474796A (en) 1991-09-04 1995-12-12 Protogene Laboratories, Inc. Method and apparatus for conducting an array of chemical reactions on a support surface
US5270170A (en) 1991-10-16 1993-12-14 Affymax Technologies N.V. Peptide library and screening method
DE59208572D1 (en) 1991-10-17 1997-07-10 Ciba Geigy Ag Bicyclic nucleosides, oligonucleotides, processes for their preparation and intermediates
FR2687679B1 (en) 1992-02-05 1994-10-28 Centre Nat Rech Scient OLIGOTHIONUCLEOTIDES.
EP0636186B1 (en) 1992-04-03 1998-11-25 The Perkin-Elmer Corporation Probe composition and method
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
GB9208733D0 (en) 1992-04-22 1992-06-10 Medical Res Council Dna sequencing method
US5257175A (en) 1992-05-08 1993-10-26 Texas Instruments Incorporated Analog control of inductive flyback voltages in a full bridge circuit
US5646020A (en) 1992-05-14 1997-07-08 Ribozyme Pharmaceuticals, Inc. Hammerhead ribozymes for preferred targets
US5247175A (en) 1992-05-27 1993-09-21 Finnigan Corporation Method and apparatus for the deconvolution of unresolved data
WO1993024834A1 (en) 1992-05-29 1993-12-09 The Rockefeller University Method and product for the sequence determination of peptides using a mass spectrometer
US5792664A (en) 1992-05-29 1998-08-11 The Rockefeller University Methods for producing and analyzing biopolymer ladders
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
EP0577558A2 (en) 1992-07-01 1994-01-05 Ciba-Geigy Ag Carbocyclic nucleosides having bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US5652355A (en) 1992-07-23 1997-07-29 Worcester Foundation For Experimental Biology Hybrid oligonucleotide phosphorothioates
EP0596205A3 (en) 1992-11-03 1996-02-21 Hewlett Packard Co Bench supervisor system.
US6194144B1 (en) 1993-01-07 2001-02-27 Sequenom, Inc. DNA sequencing by mass spectrometry
US5354934A (en) 1993-02-04 1994-10-11 Amgen Inc. Pulmonary administration of erythropoietin
GB9304618D0 (en) 1993-03-06 1993-04-21 Ciba Geigy Ag Chemical compounds
US5593826A (en) 1993-03-22 1997-01-14 Perkin-Elmer Corporation, Applied Biosystems, Inc. Enzymatic ligation of 3'amino-substituted oligonucleotides
ATE155467T1 (en) 1993-03-30 1997-08-15 Sanofi Sa ACYCLIC NUCLEOSIDE ANALOGUES AND OLIGONUCLEOTIDE SEQUENCES CONTAINING THEM
JPH08508491A (en) 1993-03-31 1996-09-10 スターリング ウインスロップ インコーポレイティド Oligonucleotides with phosphodiester bonds replaced by amide bonds
DE4311944A1 (en) 1993-04-10 1994-10-13 Degussa Coated sodium percarbonate particles, process for their preparation and detergent, cleaning and bleaching compositions containing them
US5363885A (en) 1993-06-02 1994-11-15 R. J. Reynolds Tobacco Company Robotic sample preparation system and method
US5858659A (en) 1995-11-29 1999-01-12 Affymetrix, Inc. Polymorphism detection
US6156501A (en) 1993-10-26 2000-12-05 Affymetrix, Inc. Arrays of modified nucleic acid probes and methods of use
US5908779A (en) 1993-12-01 1999-06-01 University Of Connecticut Targeted RNA degradation using nuclear antisense RNA
JP3585238B2 (en) 1993-12-09 2004-11-04 トーマス ジェファーソン ユニバーシティー Compounds and methods for site-directed mutagenesis in eukaryotic cells
US5446137B1 (en) 1993-12-09 1998-10-06 Behringwerke Ag Oligonucleotides containing 4'-substituted nucleotides
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
EP0754240B1 (en) 1994-02-07 2003-08-20 Beckman Coulter, Inc. Ligase/polymerase-mediated genetic bit analysis of single nucleotide polymorphisms and its use in genetic analysis
US6017693A (en) 1994-03-14 2000-01-25 University Of Washington Identification of nucleotides, amino acids, or carbohydrates by mass spectrometry
US5627053A (en) 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5807522A (en) 1994-06-17 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods for fabricating microarrays of biological samples
US5834189A (en) 1994-07-08 1998-11-10 Visible Genetics Inc. Method for evaluation of polymorphic genetic sequences, and the use thereof in identification of HLA types
US5597909A (en) 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US5786464C1 (en) 1994-09-19 2012-04-24 Gen Hospital Corp Overexpression of mammalian and viral proteins
US5807693A (en) 1994-11-23 1998-09-15 Icos Corporation Calcineurin inhibitory compounds and anchoring protein
US5871945A (en) 1994-11-23 1999-02-16 Icos Corporation Modulators of anchoring protein function
US5807718A (en) 1994-12-02 1998-09-15 The Scripps Research Institute Enzymatic DNA molecules
DE19515552A1 (en) 1995-04-27 1996-10-31 Europ Lab Molekularbiolog Simultaneous sequencing of nucleic acids
US5869451A (en) 1995-06-07 1999-02-09 Glaxo Group Limited Peptides and compounds that bind to a receptor
US5571676A (en) 1995-06-07 1996-11-05 Ig Laboratories, Inc. Method for mismatch-directed in vitro DNA sequencing
US6020122A (en) 1995-06-07 2000-02-01 Abbott Laboratories Hepatitis C virus second envelope (HCV-E2) glycoprotein expression system
US5981186A (en) 1995-06-30 1999-11-09 Visible Genetics, Inc. Method and apparatus for DNA-sequencing using reduced number of sequencing mixtures
US5652356A (en) 1995-08-17 1997-07-29 Hybridon, Inc. Inverted chimeric and hybrid oligonucleotides
US5869242A (en) 1995-09-18 1999-02-09 Myriad Genetics, Inc. Mass spectrometry to assess DNA sequence polymorphisms
US6190865B1 (en) 1995-09-27 2001-02-20 Epicentre Technologies Corporation Method for characterizing nucleic acid molecules
AU2069597A (en) * 1996-03-04 1997-09-22 Genetrace Systems, Inc. Methods of screening nucleic acids using mass spectrometry
DE19613082C2 (en) * 1996-04-02 1999-10-21 Koenig & Bauer Ag Method and device for the qualitative assessment of processed material
FR2749662B1 (en) 1996-06-11 1998-08-28 Elf Aquitaine ROBOTIZED LABORATORY OF SAMPLES ANALYSIS
CA2258511A1 (en) 1996-06-14 1997-12-18 Sarnoff Corporation Method for polynucleotide sequencing
CA2255839A1 (en) 1996-07-05 1998-01-15 Mark Gross Automated sample processing system
GB9618960D0 (en) 1996-09-11 1996-10-23 Medical Science Sys Inc Proteases
US6114148C1 (en) 1996-09-20 2012-05-01 Gen Hospital Corp High level expression of proteins
US6028925A (en) * 1996-09-23 2000-02-22 Rockwell International Corp. Telephonic switching system, telephonic switch and method for servicing telephone calls using virtual memory spaces
US5864137A (en) * 1996-10-01 1999-01-26 Genetrace Systems, Inc. Mass spectrometer
ATE375403T1 (en) * 1996-11-06 2007-10-15 Sequenom Inc DNA DIAGNOSTICS USING MASS SPECTROMETRY
US6017702A (en) 1996-12-05 2000-01-25 The Perkin-Elmer Corporation Chain-termination type nucleic acid sequencing method including 2'-deoxyuridine-5'-triphosphate
US5876934A (en) 1996-12-18 1999-03-02 Pharmacia Biotech Inc. DNA sequencing method
US6046005A (en) 1997-01-15 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group
US5817566A (en) 1997-03-03 1998-10-06 Taiwan Semiconductor Manufacturing Company, Ltd. Trench filling method employing oxygen densified gap filling silicon oxide layer formed with low ozone concentration
US6117634A (en) 1997-03-05 2000-09-12 The Reagents Of The University Of Michigan Nucleic acid sequencing and mapping
US6133502A (en) 1997-03-10 2000-10-17 Takeda Chemical Industries, Ltd. Monocyte chemoattractant protein and its receptor transgenic animal
US6180849B1 (en) 1997-03-17 2001-01-30 Dana-Farber Cancer Institute, Inc. Trangenic mice with a disruption in the tiar gene
US5928806A (en) * 1997-05-07 1999-07-27 Olah; George A. Recycling of carbon dioxide into methyl alcohol and related oxygenates for hydrocarbons
US5985214A (en) 1997-05-16 1999-11-16 Aurora Biosciences Corporation Systems and methods for rapidly identifying useful chemicals in liquid samples
US6011013A (en) 1997-06-20 2000-01-04 Oregon Health Sciences University Contraceptive compositions and methods
US6030778A (en) 1997-07-10 2000-02-29 Millennium Pharmaceuticals, Inc. Diagnostic assays and kits for body mass disorders associated with a polymorphism in an intron sequence of the SR-BI gene
US6112161A (en) 1997-09-17 2000-08-29 Hewlett-Packard Method, apparatus, and article of manufacture for enhanced intergration of signals
WO1999014375A2 (en) 1997-09-19 1999-03-25 Genetrace Systems, Inc. Dna typing by mass spectrometry with polymorphic dna repeat markers
US6175057B1 (en) 1997-10-08 2001-01-16 The Regents Of The University Of California Transgenic mouse model of alzheimer's disease and cerebral amyloid angiopathy
US5928952A (en) 1997-11-05 1999-07-27 Zymark Corporation Scheduled system and method for processing chemical products
DE19754482A1 (en) * 1997-11-27 1999-07-01 Epigenomics Gmbh Process for making complex DNA methylation fingerprints
US5998143A (en) 1997-12-05 1999-12-07 The Perkin-Elmer Corporation Cycle sequencing thermal profiles
CA2313380C (en) * 1997-12-08 2008-12-30 California Institute Of Technology Method for creating polynucleotide and polypeptide sequences
JP3575295B2 (en) 1998-04-15 2004-10-13 住友電装株式会社 Electrical connector plug
US6723564B2 (en) * 1998-05-07 2004-04-20 Sequenom, Inc. IR MALDI mass spectrometry of nucleic acids using liquid matrices
US6104028A (en) 1998-05-29 2000-08-15 Genetrace Systems Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry
US6294328B1 (en) 1998-06-24 2001-09-25 The Institute For Genomic Research DNA sequences for strain analysis in Mycobacterium tuberculosis
US5869275A (en) 1998-07-20 1999-02-09 Huang; Eric Z. Affinity ultrafiltration assay for transferase activity
US6132685A (en) 1998-08-10 2000-10-17 Caliper Technologies Corporation High throughput microfluidic systems and methods
US6262334B1 (en) 1998-08-31 2001-07-17 Bayer Corporation Human genes and expression products: II
US6440705B1 (en) * 1998-10-01 2002-08-27 Vincent P. Stanton, Jr. Method for analyzing polynucleotides
US6147344A (en) 1998-10-15 2000-11-14 Neogenesis, Inc Method for identifying compounds in a chemical mixture
US6331427B1 (en) 1999-03-26 2001-12-18 Millennium Pharmaceuticals, Inc. Protease homologs
US20020009394A1 (en) 1999-04-02 2002-01-24 Hubert Koster Automated process line
US7332275B2 (en) 1999-10-13 2008-02-19 Sequenom, Inc. Methods for detecting methylated nucleotides
US20030207297A1 (en) 1999-10-13 2003-11-06 Hubert Koster Methods for generating databases and databases for identifying polymorphic genetic markers
DE10021581B4 (en) 2000-04-27 2005-01-13 Auergesellschaft Gmbh Volume control for fan filter units
US6958214B2 (en) 2000-07-10 2005-10-25 Sequenom, Inc. Polymorphic kinase anchor proteins and nucleic acids encoding the same
US20030027169A1 (en) 2000-10-27 2003-02-06 Sheng Zhang One-well assay for high throughput detection of single nucleotide polymorphisms
US6548477B1 (en) 2000-11-01 2003-04-15 Praecis Pharmaceuticals Inc. Therapeutic agents and methods of use thereof for the modulation of angiogenesis
JP4212356B2 (en) 2000-11-01 2009-01-21 プラエシス ファーマシューティカルズ インク. Therapeutic agents for modulating angiogenesis and methods of use thereof
DE10112515B4 (en) * 2001-03-09 2004-02-12 Epigenomics Ag Method for the detection of cytosine methylation patterns with high sensitivity
US6522477B2 (en) * 2001-04-17 2003-02-18 Karl Storz Imaging, Inc. Endoscopic video camera with magnetic drive focusing
US20020155587A1 (en) 2001-04-20 2002-10-24 Sequenom, Inc. System and method for testing a biological sample
US6893227B2 (en) 2002-03-21 2005-05-17 Kendro Laboratory Products, Inc. Device for prevention of backward operation of scroll compressors
JP2003280586A (en) 2002-03-26 2003-10-02 Univ Toyama Organic el element and driving method therefor
US7364897B2 (en) * 2002-04-11 2008-04-29 Sequenom, Inc. Methods and devices for performing chemical reactions on a solid support
WO2003093296A2 (en) 2002-05-03 2003-11-13 Sequenom, Inc. Kinase anchor protein muteins, peptides thereof, and related methods
AU2003298733B2 (en) * 2002-11-27 2009-06-18 Agena Bioscience, Inc. Fragmentation-based methods and systems for sequence variation detection and discovery
US20070141570A1 (en) 2003-03-07 2007-06-21 Sequenom, Inc. Association of polymorphic kinase anchor proteins with cardiac phenotypes and related methods
US20050009053A1 (en) * 2003-04-25 2005-01-13 Sebastian Boecker Fragmentation-based methods and systems for de novo sequencing
DE602004019941D1 (en) 2003-07-31 2009-04-23 Sequenom Inc METHOD FOR MULTIPLEX POLYMERASE CHAIN REACTIONS AT HIGH LEVEL AND HOMOGENEOUS MASS EXTENSION REACTIONS FOR GENOTYPIZING POLYMORPHISMS
WO2005024068A2 (en) * 2003-09-05 2005-03-17 Sequenom, Inc. Allele-specific sequence variation analysis
CA2580070A1 (en) * 2004-09-10 2006-03-23 Sequenom, Inc. Methods for long-range sequence analysis of nucleic acids

Patent Citations (75)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US42112A (en) * 1864-03-29 Improvement in grain-drills
US155587A (en) * 1874-10-06 Improvement in billiard-cushions
US9394A (en) * 1852-11-09 Brick-machnsfe
US4683202B1 (en) * 1985-03-28 1990-11-27 Cetus Corp
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (en) * 1986-01-30 1990-11-27 Cetus Corp
US4826360A (en) * 1986-03-10 1989-05-02 Shimizu Construction Co., Ltd. Transfer system in a clean room
US4851018A (en) * 1986-11-28 1989-07-25 Commissariat A L'energie Atomique Installation for the storage and transfer of objects in a very clean atmosphere
US5118937A (en) * 1989-08-22 1992-06-02 Finnigan Mat Gmbh Process and device for the laser desorption of an analyte molecular ions, especially of biomolecules
US5436150A (en) * 1992-04-03 1995-07-25 The Johns Hopkins University Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease
US5440119A (en) * 1992-06-02 1995-08-08 Labowsky; Michael J. Method for eliminating noise and artifact peaks in the deconvolution of multiply charged mass spectra
US5635713A (en) * 1992-06-02 1997-06-03 Labowsky; Michael J. Method for eliminating noise and artifact the deconvolution of multiply charged mass spectra
US5506137A (en) * 1992-07-23 1996-04-09 Stratagene Purified thermostable Pyrococcus furiosus DNA ligase
US5700672A (en) * 1992-07-23 1997-12-23 Stratagene Purified thermostable pyrococcus furiousus DNA ligase
US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids
US5795714A (en) * 1992-11-06 1998-08-18 Trustees Of Boston University Method for replicating an array of nucleic acid probes
US5631134A (en) * 1992-11-06 1997-05-20 The Trustees Of Boston University Methods of preparing probe array by hybridation
US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization
US5871911A (en) * 1992-12-07 1999-02-16 Wisconsin Alumni Research Foundation Method of site-specific nucleic acid cleavage
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5691141A (en) * 1993-01-07 1997-11-25 Sequenom, Inc. DNA sequencing by mass spectrometry
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
US6225450B1 (en) * 1993-01-07 2001-05-01 Sequenom, Inc. DNA sequencing by mass spectrometry
US5872003A (en) * 1993-03-19 1999-02-16 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6074823A (en) * 1993-03-19 2000-06-13 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5622824A (en) * 1993-03-19 1997-04-22 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5851765A (en) * 1993-03-19 1998-12-22 Sequenon, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5604098A (en) * 1993-03-24 1997-02-18 Molecular Biology Resources, Inc. Methods and materials for restriction endonuclease applications
US5536649A (en) * 1993-05-11 1996-07-16 Becton, Dickinson And Company Decontamination of nucleic acid amplification reactions using uracil-N-glycosylase (UDG)
US5837832A (en) * 1993-06-25 1998-11-17 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips
US5714330A (en) * 1994-04-04 1998-02-03 Lynx Therapeutics, Inc. DNA sequencing by stepwise ligation and cleavage
US5498545A (en) * 1994-07-21 1996-03-12 Vestal; Marvin L. Mass spectrometer system and method for matrix-assisted laser desorption measurements
US5453613A (en) * 1994-10-21 1995-09-26 Hewlett Packard Company Mass spectra interpretation system including spectra extraction
US6589485B2 (en) * 1995-03-17 2003-07-08 Sequenom, Inc. Solid support for mass spectrometry
US6277573B1 (en) * 1995-03-17 2001-08-21 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6043031A (en) * 1995-03-17 2000-03-28 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6300076B1 (en) * 1995-03-17 2001-10-09 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6602662B1 (en) * 1995-03-17 2003-08-05 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6428955B1 (en) * 1995-03-17 2002-08-06 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US5874283A (en) * 1995-05-30 1999-02-23 John Joseph Harrington Mammalian flap-specific endonuclease
US5858705A (en) * 1995-06-05 1999-01-12 Human Genome Sciences, Inc. Polynucleotides encoding human DNA ligase III and methods of using these polynucleotides
US5853979A (en) * 1995-06-30 1998-12-29 Visible Genetics Inc. Method and system for DNA sequence determination and mutation detection with reference to a standard
US5952176A (en) * 1995-07-11 1999-09-14 Forfas (Trading As Bioresearch Ireland) Glycosylase mediated detection of nucleotide sequences at candidate loci
US6146854A (en) * 1995-08-31 2000-11-14 Sequenom, Inc. Filtration processes, kits and devices for isolating plasmids
US6090606A (en) * 1996-01-24 2000-07-18 Third Wave Technologies, Inc. Cleavage agents
US5843669A (en) * 1996-01-24 1998-12-01 Third Wave Technologies, Inc. Cleavage of nucleic acid acid using thermostable methoanococcus jannaschii FEN-1 endonucleases
US5686656A (en) * 1996-02-27 1997-11-11 Aviv Amirav Method and device for the introduction of a sample into a gas chromatograph
US5928906A (en) * 1996-05-09 1999-07-27 Sequenom, Inc. Process for direct sequencing during template amplification
US6022688A (en) * 1996-05-13 2000-02-08 Sequenom, Inc. Method for dissociating biotin complexes
US6200756B1 (en) * 1996-06-03 2001-03-13 The Johns Hopkins University School Of Medicine Methods for identifying methylation patterns in a CpG-containing nucleic acid
US6017704A (en) * 1996-06-03 2000-01-25 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US5786146A (en) * 1996-06-03 1998-07-28 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US6265171B1 (en) * 1996-06-03 2001-07-24 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguish modified methylated and non-methylated nucleic acids
US5885841A (en) * 1996-09-11 1999-03-23 Eli Lilly And Company System and methods for qualitatively and quantitatively comparing complex admixtures using single ion chromatograms derived from spectroscopic analysis of such admixtures
US6423966B2 (en) * 1996-09-19 2002-07-23 Sequenom, Inc. Method and apparatus for maldi analysis
US6111251A (en) * 1996-09-19 2000-08-29 Sequenom, Inc. Method and apparatus for MALDI analysis
US5777324A (en) * 1996-09-19 1998-07-07 Sequenom, Inc. Method and apparatus for maldi analysis
US6566055B1 (en) * 1996-09-19 2003-05-20 Sequenom, Inc. Methods of preparing nucleic acids for mass spectrometric analysis
US5900481A (en) * 1996-11-06 1999-05-04 Sequenom, Inc. Bead linkers for immobilizing nucleic acids to solid supports
US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6133436A (en) * 1996-11-06 2000-10-17 Sequenom, Inc. Beads bound to a solid support and to nucleic acids
US6024925A (en) * 1997-01-23 2000-02-15 Sequenom, Inc. Systems and methods for preparing low volume analyte array elements
US6059724A (en) * 1997-02-14 2000-05-09 Biosignal, Inc. System for predicting future health
US5928870A (en) * 1997-06-16 1999-07-27 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US5976806A (en) * 1997-06-25 1999-11-02 Pioneer Hi-Bred International, Inc. DNA ligase assay
US5975492A (en) * 1997-07-14 1999-11-02 Brenes; Arthur Bellows driver slot valve
US6322970B1 (en) * 1997-09-02 2001-11-27 Sequenom, Inc. Mass spectrometric detection of polypeptides
US6207370B1 (en) * 1997-09-02 2001-03-27 Sequenom, Inc. Diagnostics based on mass spectrometric detection of translated target polypeptides
US5888795A (en) * 1997-09-09 1999-03-30 Becton, Dickinson And Company Thermostable uracil DNA glycosylase and methods of use
US6268131B1 (en) * 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids
US6188064B1 (en) * 1998-01-29 2001-02-13 Bruker Daltonik Gmbh Mass spectrometry method for accurate mass determination of unknown ions
US6054276A (en) * 1998-02-23 2000-04-25 Macevicz; Stephen C. DNA restriction site mapping
US6099553A (en) * 1998-05-21 2000-08-08 Applied Medical Resources Corporation Suture clinch
US6270835B1 (en) * 1999-10-07 2001-08-07 Microcoating Technologies, Inc. Formation of this film capacitors

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7759065B2 (en) 1995-03-17 2010-07-20 Sequenom, Inc. Mass spectrometric methods for detecting mutations in a target nucleic acid
US8821816B2 (en) 1997-01-23 2014-09-02 Agena Biosciences, Inc. Matrix-assisted laser desorption ionization mass spectrometry substrates having low volume matrix array elements
US20030096426A1 (en) * 1997-01-23 2003-05-22 Daniel P. Little Systems and methods for preparing and analyzing low volume analyte array elements
US7668658B2 (en) 1999-10-13 2010-02-23 Sequenom, Inc. Methods for generating databases and databases for identifying polymorphic genetic markers
US8818735B2 (en) 1999-10-13 2014-08-26 Sequenom, Inc. Methods for generating databases and databases for identifying polymorphic genetic markers
US8229677B2 (en) 1999-10-13 2012-07-24 Sequenom, Inc. Methods for generating databases and databases for identifying polymorphic genetic markers
US9669376B2 (en) 2000-10-30 2017-06-06 Agena Bioscience, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate
US8999266B2 (en) 2000-10-30 2015-04-07 Agena Bioscience, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate
US7820378B2 (en) 2002-11-27 2010-10-26 Sequenom, Inc. Fragmentation-based methods and systems for sequence variation detection and discovery
US7468248B2 (en) 2002-12-31 2008-12-23 Cargill, Incorporated Methods and systems for inferring bovine traits
US8026064B2 (en) 2002-12-31 2011-09-27 Metamorphix, Inc. Compositions, methods and systems for inferring bovine breed
US20090221432A1 (en) * 2002-12-31 2009-09-03 Denise Sue K Compositions, methods and systems for inferring bovine breed
US20080268454A1 (en) * 2002-12-31 2008-10-30 Denise Sue K Compositions, methods and systems for inferring bovine breed or trait
US7709206B2 (en) 2002-12-31 2010-05-04 Metamorphix, Inc. Compositions, methods and systems for inferring bovine breed or trait
US11053547B2 (en) 2002-12-31 2021-07-06 Branhaven LLC Methods and systems for inferring bovine traits
US7511127B2 (en) 2002-12-31 2009-03-31 Cargill, Incorporated Compositions, methods and systems for inferring bovine breed
US20070031845A1 (en) * 2002-12-31 2007-02-08 Mmi Genomics, Inc. Compositions, methods and systems for inferring bovine breed
US9206478B2 (en) 2002-12-31 2015-12-08 Branhaven LLC Methods and systems for inferring bovine traits
US10190167B2 (en) 2002-12-31 2019-01-29 Branhaven LLC Methods and systems for inferring bovine traits
US9982311B2 (en) 2002-12-31 2018-05-29 Branhaven LLC Compositions, methods, and systems for inferring bovine breed
US8450064B2 (en) 2002-12-31 2013-05-28 Cargill Incorporated Methods and systems for inferring bovine traits
US8669056B2 (en) 2002-12-31 2014-03-11 Cargill Incorporated Compositions, methods, and systems for inferring bovine breed
US20050287531A1 (en) * 2002-12-31 2005-12-29 Mmi Genomics, Inc. Methods and systems for inferring bovine traits
US20050260603A1 (en) * 2002-12-31 2005-11-24 Mmi Genomics, Inc. Compositions for inferring bovine traits
US20070112585A1 (en) * 2003-08-01 2007-05-17 Breiter Hans C Cognition analysis
WO2005020788A2 (en) * 2003-08-01 2005-03-10 The General Hospital Corporation Cognition analysis
WO2005020788A3 (en) * 2003-08-01 2006-06-29 Gen Hospital Corp Cognition analysis
US9394565B2 (en) 2003-09-05 2016-07-19 Agena Bioscience, Inc. Allele-specific sequence variation analysis
US20100162423A1 (en) * 2003-10-24 2010-06-24 Metamorphix, Inc. Methods and Systems for Inferring Traits to Breed and Manage Non-Beef Livestock
WO2005098050A2 (en) 2004-03-26 2005-10-20 Sequenom, Inc. Base specific cleavage of methylation-specific amplification products in combination with mass analysis
US9249456B2 (en) 2004-03-26 2016-02-02 Agena Bioscience, Inc. Base specific cleavage of methylation-specific amplification products in combination with mass analysis
EP2395098A1 (en) 2004-03-26 2011-12-14 Sequenom, Inc. Base specific cleavage of methylation-specific amplification products in combination with mass analysis
US9068953B2 (en) 2007-09-17 2015-06-30 Agena Bioscience, Inc. Integrated robotic sample transfer device
US20210017592A1 (en) * 2017-07-07 2021-01-21 Massachusetts Institute Of Technology Systems and methods for genetic identification and analysis
US11655498B2 (en) * 2017-07-07 2023-05-23 Massachusetts Institute Of Technology Systems and methods for genetic identification and analysis

Also Published As

Publication number Publication date
US20100292930A1 (en) 2010-11-18
US7332275B2 (en) 2008-02-19
US7668658B2 (en) 2010-02-23
US8818735B2 (en) 2014-08-26
US20150005194A1 (en) 2015-01-01
US20030180748A1 (en) 2003-09-25
US20120301882A1 (en) 2012-11-29
US8229677B2 (en) 2012-07-24
US20030180749A1 (en) 2003-09-25

Similar Documents

Publication Publication Date Title
AU776811B2 (en) Methods for generating databases and databases for identifying polymorphic genetic markers
US20030190644A1 (en) Methods for generating databases and databases for identifying polymorphic genetic markers
CA2414495A1 (en) Polymorphic kinase anchor proteins and nucleic acids encoding the same
AU781437B2 (en) A novel BAP28 gene and protein
US20040014067A1 (en) Amplification methods and compositions
WO2004033649A2 (en) High throughput multiplex dna sequence amplifications
AU779411B2 (en) Biallelic markers derived from genomic regions carrying genes involved in arachidonic acid metabolism
KR20150043566A (en) Use of markers in the identification of cardiotoxic agents
CA2514187A1 (en) Expression profiles for colon cancer and methods of use
CA2442820A1 (en) Microarray gene expression profiling in clear cell renal cell carcinoma: prognosis and drug target identification
KR101157526B1 (en) Snp for diagnosing adhd, microarray and kit comprising the same, and method of diagnosing adhd using thereof
KR101141543B1 (en) Polynucleotides derived from ALDH4A1, PINK1, DDOST, KIF17, LMX1A, SRGAP2, ASB3, PSME4, ANXA4, GMCL1, and MAP2 genes comprising single nucleotide polymorphisms, microarrays and diagnostic kits comprising the same, and analytic methods using the same
JP2003144176A (en) Detection method for gene polymorphism
US20030207297A1 (en) Methods for generating databases and databases for identifying polymorphic genetic markers
US20030235847A1 (en) Association of polymorphisms in the SOST gene region with bone mineral density
US20040170974A1 (en) Detection of polymorphisms in the human 5-lipoxygenase gene
US6692909B1 (en) Coding sequence polymorphisms in vascular pathology genes
AU784761B2 (en) Biallelic markers related to genes involved in drug metabolism
CN106086018A (en) NR_047662.2 and the reagent of vitro detection, preparation or test kit, application, detection method
CA2518238A1 (en) Association of polymorphic kinase anchor proteins with cardiac phenotypes and related methods
TWI358456B (en) Method of determining susceptibility of high myopi
US20030215819A1 (en) Compositions and methods for inferring a response to statin
CN113151447A (en) Capture probe and kit for primary atopic disease related gene and application thereof
KR102250063B1 (en) Method for identifying causative genes of tourette syndrome
CN100516876C (en) Methods for diagnosing RCC and other solid tumors

Legal Events

Date Code Title Description
AS Assignment

Owner name: SEQUENOM, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KOSTER, HUBERT;BRAUN, ANDREAS;VAN DEN BOOM, DIRK;AND OTHERS;REEL/FRAME:013823/0001;SIGNING DATES FROM 20001211 TO 20010122

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION