WO1998000708A1 - Method and apparatus for alignment of signals for use in dna base-calling - Google Patents
Method and apparatus for alignment of signals for use in dna base-calling Download PDFInfo
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- WO1998000708A1 WO1998000708A1 PCT/CA1997/000463 CA9700463W WO9800708A1 WO 1998000708 A1 WO1998000708 A1 WO 1998000708A1 CA 9700463 W CA9700463 W CA 9700463W WO 9800708 A1 WO9800708 A1 WO 9800708A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B30/10—Sequence alignment; Homology search
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06F—ELECTRIC DIGITAL DATA PROCESSING
- G06F2218/00—Aspects of pattern recognition specially adapted for signal processing
- G06F2218/12—Classification; Matching
- G06F2218/14—Classification; Matching by matching peak patterns
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
Definitions
- This invention relates to a method of processing output signals from an automated eiectrophoresis detection apparatus, and to an apparatus which employs this method for sequencing nucleic acids.
- One of the steps in nucleotide sequence determination of a subject nucleic acid molecule is interpretation of the pattern of nucleic acid fragments which results from electro- phoretic separation of fragments, or reaction products, of a DNA sequencing reaction (the "fragment pattern").
- the interpretation colloquially known as “base calling”, involves determination from the recorded fragment pattern of the order of four nucleotide bases, A (adenine), C (cytosine), G (guanine) and T (thymine) for DNA or U (uracil) for RN A in the subject nucleic acid molecule.
- an automated eiectrophoresis detection apparatus can be used to detect the passage of migrating bands in real time.
- Applied Biosystems, Inc. Fluorescence-Activated Cell Sorting
- Pharmacia Biotech, Inc. Pancataway, NJ
- a reliable method of aligning data that can produce data which takes into account non-linear shifting and stretching of signal output, is highly desirable particularly for high-speed DNA sequencing.
- the aligned data set may be displayed on a video screen of a sequencing apparatus, or may be used as the data set for a base-calling process.
- Fig. 1 shows the alignment process of the invention in flow chart form
- Fig. 2 illustrates a preliminary (unaligned) signal recorded from an automated DNA sequencing apparatus over 15 seconds
- Fig. 3 illustrates the detection of peaks from a preliminary signal
- Fig. 4 illustrates a normalized data stream
- Figs. 5A and 5B illustrate a normalization method employed for peaks and valleys
- Figs. 6A and 6B illustrate the preliminary alignment of four normalized data streams representing time based alignment of each of the A, C, G and T output channels;
- Fig. 7 illustrates alignment of the 4 normalized data streams with respect to minimization of the "cost" function
- Figs. 8A and 8B shows exemplary warp functions for data points in the C, G and T traces relative to the A trace;
- Fig. 9 illustrates a method for determining a standard gaussian peak width
- Fig 10 shows an example of the classification of singletons using the features of peaks in the detected signal
- Fig. 1 1 illustrates a base-calling method useful with the aligned data traces
- Fig. 12 shows an apparatus in accordance with the invention
- the present invention provides a method of aligning data traces which can be used to align data traces from an automated eiectrophoresis detection apparatus for use in base- calling.
- an optimization routine such as a simulated annealing algorithm is used to determine coefficients of stretch and shift to align data traces, each representing one of the four chain-termination DNA sequencing reactions
- base-cal ng may proceed with a high degree of accuracy and repeatability.
- Data trace refers to the series of peaks and valleys representing the migrating bands of o gonucleotide fragments produced in one chain termination sequencing reaction and detected in a DNA sequencer Such data traces are sometimes referred to as sequence chromatograms or a chromatographic trace
- the data trace may be either a raw data trace or a "conditioned" data trace
- “Shift” as used in the specification and claims of this application is interchangeable with “offset” and refers to the number of data points which the signal output is displaced, either positively or negatively from its original position
- “Stretch” as used in the specification and claims of this application refers to increase or decrease in spacing between data points of a signal output relative to the original spacing. The stretch may be constant across a segment of data or may follow a second or higher order polynomial
- a "warp function" as used in the specification and claims of this application is an instantaneous representation of optimized shift and stretch at each data point
- the warp function can be represented graphically either as a data point- for-data point plot for one data trace versus a standard or second data trace, or as a plot of displacement as a function of data point number.
- Standard traces may be a trace representing an average of multiple experimental runs, a base separation function, or a standard derived from a text sequence.
- the wa ⁇ function is applied to a data trace to obtain a "wa ⁇ ed data trace.”
- Fig. 1 shows the alignment process of the invention in flow chart form. As shown, the first step in the method is the identification of peaks in each of several (generally four) data traces.
- these traces are next normalized to a constant height. This step may be unnecessary if the DNA Sequencer and chemistry used produce substantially uniform peak sizes (homozygous peak heights within about 10 %) or if the traces are nearly aligned from the sequencer.
- the term "normalized data traces" refers to data traces having this level of consistency, whether as a result of normalization step or as a result of sufficient initial uniformity. Normalization may also be unnecessary, even for non-uniform peak sizes, if the cost function used in the alignment is independent of peak height and area.
- the data traces are then combined in an initial alignment and divided into a plurality of segments or windows.
- optimized stretch and shift values are determined using an iterative optimization routine which converges towards a "best" solution for alignment of the data traces.
- the quality of the various trial alignment is assessed for comparison pu ⁇ oses using a cost function.
- cost functions can be employed including ( 1 ) a cost function which minimizes overlap of the data traces combined within the segment; and (2) a cost function which maximizes the regularity of peak distribution the segment.
- the shift and stretch values are used to define wa ⁇ functions. These wa ⁇ functions are applied to the individual data traces to produce wa ⁇ ed data traces which are assembled as aligned data useful for base-calling.
- FIG. 2 shows a typical raw data trace for one of the four chain-termination DNA sequencing reactions, as detected on a Visible Genetics Inc.
- the X-axis represents time while the Y axis represents fluorescence detection.
- the data trace reveals a series of bands of fluorescent molecules passing through the detection site, as expected from a typical chain-termination DNA sequencing reaction.
- Fig. 2 shows several of the features which complicate the use of raw data for base- calling, and illustrates the need for the present invention to provide appropriately aligned signals for this pu ⁇ ose.
- the fluorescence intensity can vary from one band to another.
- the present invention provides a method for converting this raw signal, and its three counte ⁇ art signals for the other three sequencing reactions, into aligned data which is highly suitable for base-calling.
- the data trace which is processed in accordance with the method of the invention is preferably a signal collected using the fluorescence detection apparatus of an automated DNA sequencer
- the present invention is applicable to any data set which reflects the separation of ohgonucleotide fragments in space or time, including real-time fragment patterns using any type of detector, for example a polarization detector as described in US Patent No
- This conditioning can be done, for example, using conventional baseline correction and noise reduction techniques to yield a "conditioned" data trace
- three methods of signal processing commonly used are background subtraction, low frequency filtration and high frequency filtration, and any of these may be used, singly or in combination to produce a conditioned signal to be used as a conditioned data trace in the method of the invention.
- the data is conditioned by background subtraction using a non-linear filter such as an erosion filter, with or without a low-pass filter to eliminate systemic noise.
- a non-linear filter such as an erosion filter
- the preferred low-pass filtration technique is non-causal gaussian convolution.
- the data trace (raw or conditioned) is searched for peaks. Peaks can be identified as the middle data point of three consecutive data points wherein the inside data point is higher than the two outside data points. Fig. 3. More sophisticated methods of peak detection are also possible. For example, a preferred method involves using the "three-point" method to segment the data trace, and then joining the segments. A trace feature is assigned as an actual peak whenever the difference between a maximum and an adjacent minimum exceeds a threshold value, e.g., 5%. A minimum peak height from the base-line may also be required to eliminate spurious peaks.
- a threshold value e.g., 5%
- the data trace is normalized so that all of the identified peaks have the same height which is assigned a common value, e.g., 1. (Fig. 4).
- This process reduces signal variations due to chemistry and enzyme function, and works effectively for homozygous samples and for many heterozygotes having moderate, i.e., less than about 5 to 10%, heterozygosity in a 200 base pair or larger region being sequenced. It will be appreciated that when the data trace has substantially uniform peak height, normalization can be omitted.
- the points between each peak are assigned a nume ⁇ cal height value based on their position in the data trace relative to a hypothetical line joining consecutive peaks and the base line of the signal.
- the valley between peaks 1 and 2 has a minimum at a point which is approximately 25% of the distance from the baseline B to the line C joining peaks 1 and 2. The minimum of this valley is therefore assigned a value of about .25. (See Fig. 5B).
- the valley between peaks 2 and 3 has a minimum at a point which is approximately 80% of the distance from the baseline B to the line D joining peaks 2 and 3. The minimum of this valley is therefore assigned a value of about 0.8 in the normalized data trace.
- the next step in the method of the invention is the determination of shift and stretch coefficients for a set of four normalized data traces, one for each sequencing reaction. This is accomplished by combining the four normalized data traces as shown diagrammatical ly in
- Figs. 6A and 6B and determining coefficients of shift and stretch for selected data points within each normalized data trace which optimize a "cost" function.
- the cost function generally reflects the suitability of a trial alignment resulting from the application of the trial stretch and shift coefficients to the combined data traces.
- One type of cost function evaluates the extent of overlap of peaks in the combined normalized data traces to which the coefficients have been applied, and is considered optimized when the extent of overlap is at a minimum. It will be understood that the terms "best solution,” “optimized” and “minimum” as used herein do not require absolute optimization to an absolute minimum, which could require unreasonably long periods of analysis time, but only require a practical level of optimization sufficient to achieve satisfactory alignment of the data traces for base-calling.
- the cost function measures the total area above the combined normalized data traces, i.e., the dotted area in Fig. 5B.
- the normalized data traces are then shifted and stretched in an effort to minimize the value of "cost.”
- "cost" is set equal to the area below the combined normalized data traces, and the data traces are then shifted and stretched to maximize the value of cost (i.e. reducing the overlap of the peaks).
- this latter approach emphasizes less valuable features of the data traces than using the area above the curves as the cost function.
- the cost function combines the total area above the combined data traces (the dotted regions in Fig. 6B) with the area below the highest edge formed by the combined data traces and above the second highest edge of the combined data traces (the lined regions in Fig. 6B).
- the cost function is at a minimum when the first of these areas is minimized and the second of these areas is maximized.
- an aligned normalized data set as shown in Fig. 7 results.
- the cost function used in the method of the invention may also compare the experimental data traces to a set of model data traces.
- a suitable cost function is the area between the experimental data trace and a corresponding data trace from a set of model data traces that have been prealigned.
- the cost function is optimized when the area between the traces is at a minimum.
- Standard sets of model data traces comprised of random distributions of bases might also be used in such a comparison, although this increases the cost function space.
- cost funclion that can be used in the method of the invention evaluates the regularity of peak spacing achieved by the trial alignment.
- cost functions do not require normalization, because they do not depend on peak height or area.
- An example of such a function would be a function determining the standard deviation of the distance between adjacent peaks, excluding obvious outliers, with a low value for the cost function being desirable.
- Another example of a cost function of this type determines the distance between each peak and the closest base position within a postulated regular array of peak spacings. The cost function is optimized when the standard deviation of these distances for all of the peaks is at a minimum.
- the postulated peak spacing in this array is tested over a several trial values established for a specific instrumental and experimental configuration, thus adding an additional dimension to the cost function space. For example, where it is found that the normal peak spacing is 7 data points, trial peak spacings of 3 to 9 data points are suitably tested for each trial alignment to determine the lowest value of the cost function.
- cost functions discussed above can be used individually or in combination since they all work towards a common goal, the optimum alignment of the data traces.
- alternative cost functions can be created as the product or dividend that results when two or more of the cost functions are combined.
- simulated annealing determines coefficients of stretch and shift for signal outputs from a Visible Genetics MicroGene BlasterTM as follows.
- the normalized data trace consists of a series of data points generated every 0.5 seconds. Each peak consists of about 6 to 7 data points, and requires 3 to 3.5 seconds to pass through the detection zone.
- the window shown represents about 90 data points or about 15 peaks. In a preferred embodiment, however, windows of 180 to 350 data points, representing about 30 to 50 peaks, most preferably of about 250 data points representing about 40 peaks are used.
- One window is created for data from each of the four data traces, and the windows would be initially aligned on the basis of data-point number. Superimposing the four windows reveals a non-minimized "cost” result, that is, the amount of "cost" area is greater than it could be.
- the parameter space for b and m is empirically selected.
- the offset coefficient (b) usually falls within 30 data points of the initial time-based alignment. Offset steps of 0.35 data points are suitably employed, thus providing a parameter space of 200 choices For MicroGene BlasterTM data, this offset represents about 5 data peaks.
- the stretch coefficient (m) usually falls within 5%. These steps are suitably made in 0.66% amounts, thus requiring
- the total range of parameters for stretch and shift for each signal output is therefore 3000 (200 * 15).
- the range of parameters for alignment of three channels with respect to the fourth channel is therefore 27 billion (3000 ).
- the coefficient are applied to the three signal output functions and the functions are stretched and/or shifted accordingly.
- the adjusted functions are superimposed with the fourth signal output and the "cost" area is recalculated. The cost will either be lower, higher or the same as before.
- the simulated annealing theory selects parameters for testing according to a variation of the Monte Carlo search technique "Boltzmann Annealing" known as Adaptive Simulated Annealing (or Very Fast Simulated Re-Annealing).
- Simulated Annealing code is generally available to those skilled in the art over the Internet at http://www.ingber.com. The code provides operational steps for rapidly searching a large parameter space for an optimal solution given a cost function.
- An explanation of Simulated Annealing is found in Ingber, A.L. "Very Fast Simulated Re-Annealing" J. Math. Comput.
- Modeling (1989) 12: 967-973 The simulated annealing technique employed in the invention uses algorithms which are well-known to those skilled in the mathematical arts in the following novel fashion. Coefficients of stretch and shift are at first randomly selected and applied to the normalized data traces. The cost is calculated. New coefficients are then selected within a range defined by the annealing schedule (or temperature function "T") which governs the amount by which coefficients may be changed with each trial. The new coefficients are applied and cost is again determined. If the value of cost is lower than before, then the new point is used as the starting point for the next calculation. If the value of cost is higher, then the original coefficients are usually used again as the starting point of the next calculation.
- T temperature function
- next window of data is analyzed.
- the next window is selected to be the same number of data points as the first window, with an overlap of about 50% with the first window.
- coefficients of shift and stretch are identified which provide the lowest cost value when applied to the signal output functions.
- coefficients are determined for a series of piecewise domains, e.g., piecewise linear or cubic domains. The process of selecting windows and calculating coefficients continues until all the data has been analyzed.
- the annealing schedule T can be much shorter, for example testing only 1500 sets of coefficients as opposed to 5000.
- subsequent windows may m fact be evaluated as two "sub-windows " In the first sub-window, stretch coefficients are constrained such that the wa ⁇ function does not change the offset already established at the center of the previous full window In the second sub-window, the stretch is allowed to vary in a narrow range.
- This function reflects the relationship between optimum-cost shift values for each window
- a curve is defined which gives coefficients of shift for each point in a data trace, and reflects stretch at each location by the slope of the curve.
- the wa ⁇ functions may be determined relative to one of the four data traces In this case, the coefficients for the one fixed data trace will all be one
- the phrase "generating a wa ⁇ function for each normalized data trace" encompasses both of these embodiments
- the wa ⁇ function may be represented by a plot of alignment of data points of the three wa ⁇ ed traces, e.g., C, G and T against A as shown in Fig 8 A or as a plot of displacement from the A trace versus data point as shown in Fig. 8B.
- Each wa ⁇ function is applied to its respective raw or conditioned data trace to adjust the alignment of the data trace and generate a "wa ⁇ ed data trace " The four wa ⁇ ed data traces are then combined in alignment to produce an aligned data set.
- An additional peak spacing wa ⁇ function may be generated and applied to adjust for variations in peak spacing as part of creating the wa ⁇ ed data traces.
- the peak spacing wa ⁇ function, and also the base separation function useable as a standard trace, are generated by
- SUBSTTTUTE SHEET (RULE 26) testing postulated peak separation values in successive windows of the data trace and minimizing a peak separation cost function for each window.
- a suitable cost function is
- d is the distance between each actual peak and a hypothetical peak located at the position fixed by the postulated peak separation value.
- Presentation of the aligned data set may be done internally within a computer for use with base-calling functions, or it may involve display of the aligned data set on a video monitor. Either way, the presentation allows further use to be made of the modified output signals, for base-calling and other pu ⁇ oses.
- the video display of aligned data may be useful to permit an operator to make manual adjustments, and to observe inaccuracies in base-calling.
- Base-calling on the aligned data set may be performed in a variety of ways, including those base-calling techniques described in Tibbetts and Bowling (US Pat. No. 5,365,455) and Dam et al (US Pat. No. 5,1 19,316). Two preferred approaches to base-calling are described in detail below.
- peaks in each wa ⁇ ed data trace making up the aligned data set are identified in the same manner in which peak detection was performed prior to normalization of the data traces.
- a minimum peak height from the base-line may be selected by the operator to avoid spurious results.
- Identified peaks are then used for base- calling. Occasionally, peaks may represent a plurality of bands.
- gaussian deconvolution whereby a peak is deconvolved into one or more standard gaussian peaks representing singleton peaks. It is found that peaks generated from DNA sequencing reactions using T7 polymerase (Pharmacia, Sweden) and Thermo SequenaseTM (Amersham Life Sciences) generate the most consistent gaussian peaks.
- the standard gaussian peak is determined as shown in Fig. 9. Peaks are located in a conditioned data trace from one channel. A line is drawn between peak points. The point on the line halfway between peaks is joined to the data trace by a line L pe ⁇ endicular to the baseline. The area under the curve A and between the two pe ⁇ endicular lines (L(x), L(x+1)) is determined. Height (h) is measured from the baseline to the peak, h and A are used to calculate sigma ( ⁇ ) according to the equation:
- ⁇ represents the distance on the x-axis between the peak and the point at which the value of the gaussian function
- ⁇ is determined. For those peaks where the length of both L(x) and L(x+1 ) are greater than l ⁇ h, a linear regression is performed on the value of ⁇ . Statistically, at most about 25% of peaks are expected to represent doubletons, triples or greater, so using a second linear regression to correct for a width trend over each window, the narrowest 50% of the peaks are selected for use in constructing a piecewise cubic "singleton width discriminant function" that specifies a model width of a singleton at each location in the data array. All peaks in the window that are narrower or equal to this function are deemed to be singletons to a first approximation. This approximation may be further refined using constraints such as peak area, etc. (Fig. 10) In this way, a standard or model singleton gaussian peak height and width at any point on the data trace may be defined.
- the characteristics of the standard gaussian peak(s) and the positions of the singletons found via the discriminant function in conjunction with the base separation function are then used to classify all the peaks in the aligned data traces.
- the features (e.g., height, width and/or area) of the standard peak are compared to the features of a detected signal peak to determine the number of bases represented.
- the characteristics of the standard gaussian peak are used to determine whether two, three or four peaks are most likely to be the number of base pairs represented by the large peak.
- the difference between the area of the large peak and the area of the standard gaussian peak can be evaluated. If the area of the large peak is approximately three times the area of the standard peak, then the large peak is treated as representing three bases. Similarly, if the area is closer to four times the area of the standard peak, the large peak is treated as representing four bases.
- a similar, although less sensitive analysis can be performed based on the height of the peaks. The preferred analysis takes multiple peak features into account.
- the primer peak may be eliminated or ignored as described for preparing the normalized signal output, so as not to interfere with base-calling.
- Another method of identifying and eliminating the primer peak uses a peak counting method.
- the data stream is divided into windows of a certain number of data points.
- the peaks in each window are counted.
- a window that normally would include 10 peaks may have only 2 peaks. This window is eliminated from consideration, and other windows are used for alignment and base-calling.
- Fig. 1 1 outlines a second approach to base-calling using the aligned data set of the invention. In this case, peaks are identified within the aligned data set, for example as discussed above, and the data set is then divided into segments or windows.
- a subset of the peaks are selected, for example 45% of the peaks, using selection criteria which select those peaks which are most likely to be "singleton peaks," i.e., peaks which represent only a single base and where that base is not the same as either adjacent base such that there should be no compression with adjacent peaks.
- the selection of singleton peaks follows a several step process in which the features of each peak, i.e., width, height and area etc. are extracted and plotted in n-space. Based on the distribution of the peaks and the fact that, statistically, 56% of the bases in any sequence should be singletons within this definition, the region of n-space most likely to contain singleton peaks is defined. The peaks which fall within this region of space are then collected. From the first pass singleton classification, piecewise singleton height and width functions that estimate model singleton features are defined for each segment or window. These functions are then used to reclassify the peaks from the aligned data set as singleton or non-singleton peaks.
- a single piecewise single base separation distance function that estimates base-to-base separation is determined and used to provide postulated positions for all bases. These positions, combined with the actual peak features, neighboring characteristics and raw signal characteristics are then used to provide a best estimate of base call, together with a measure of the certainty of that call at each postulated base positions.
- the method of the invention is advantageously practiced using a dedicated apparatus for determining nucleic acid sequences. As depicted in Fig.
- such an apparatus comprises a sequencer 90 having an eiectrophoresis gel holder 901 disposed between electrodes 902 which are used to apply an electric field to a gel placed in the holder to cause ohgonucleotide fragments to migrate within the gel; a detection system comprising a source 908 for an interrogating beam 92 and a detector 907 for detecting the passage of ohgonucleotide fragments through a detection zone, for example by monitoring emitted light 99; and a data processing system 96 operatively connected to the detector 95 for receiving raw data traces for each of the four chain termination product mixtures for a sample.
- Suitable gel holders, electrodes and detection systems are disclosed in US Patent Application No. 08/353.932, PCT Patent Application No. PCT/US95/15951 , and US Patent Application No. US Patent
- the data processing system is suitably a personal or mini-computer which has stored therein a programmed instruction set effective to identify peaks in each of the four data traces; normalize the height of said peaks in each of said data traces to a common value to generate four normalized data traces; combine the four normalized data traces in an initial alignment; determine coefficients of shift and stretch for selected data points within each normalized data trace, said coefficients optimizing a cost function which reflects the extent of overlap of peaks in combined normalized data traces to which the coefficients have been applied, said cost function being optimized when the extent of overlap is at a minimum; generate a wa ⁇ function for each normalized data trace from the coefficients of shift and stretch determined for the selected data points; a PP'y eacn war P functions to the respective data trace or the normalized data trace to produce four wa ⁇ ed data traces; and align the four wa ⁇ ed data traces to form an aligned data set.
- the data processing system may be connected to a video display 97 for displaying the align
- the apparatus of the invention reports confidence levels to the system operator for some or all of the bases identified in the sequence.
- the confidence level advantageously reflects both ( 1 ) the arithmetic agreement between the signal and the model, and (2) other features of the data signal (for example expanded peak width) which may indicate reasons that the confidence level should be lower than the apparent level based on arithmetic agreements.
- This confidence level can be reported for all peaks, or it can be reported only for those peaks for which the confidence level falls below a selected threshold value. Peaks may also be flagged during the reporting process to report ambiguities in the identification of the number of bases represented by a multiple peak feature.
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Cited By (22)
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WO2000000637A2 (en) * | 1998-06-26 | 2000-01-06 | Visible Genetics Inc. | Method for sequencing nucleic acids with reduced errors |
WO2000072218A2 (en) * | 1999-05-25 | 2000-11-30 | Digital Gene Technologies, Inc. | Methods for normalization of experimental data |
WO2000072182A2 (en) * | 1999-05-25 | 2000-11-30 | Digital Gene Technologies, Inc. | Methods and system for amplitude normalization and selection of data peaks |
US6303303B1 (en) | 1995-06-30 | 2001-10-16 | Visible Genetics Inc | Method and system for DNA sequence determination and mutation detection |
GB2362699A (en) * | 2000-05-24 | 2001-11-28 | Cartercopters Llc | A landing gear shock absorber with a variable viscosity fluid |
WO2002015107A2 (en) * | 2000-08-14 | 2002-02-21 | Incyte Genomics, Inc. | Basecalling system and protocol |
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US5916747A (en) | 1999-06-29 |
AU3250797A (en) | 1998-01-21 |
CA2259314A1 (en) | 1998-01-08 |
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