Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-02
2001-08-14
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S912000, C536S024310, C536S024330, C204S450000, C204S461000, C204S466000, C204S600000, C204S612000, C702S019000, C702S020000, C382S128000, C382S129000
Reexamination Certificate
active
06274317
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to automated allele callers for use in identifying alleles for genetic markers.
BACKGROUND
Genotyping is an important technique in genetic research for mapping a genome and localizing genes that are linked to inherited characteristics such as genetic diseases. In genetic disease studies, for example, a library of genetic markers is screened against DNA samples from affected individuals and their families. Resulting data are analyzed to find chromosomal regions whose transmission from parent to child is correlated with (i.e., linked to) transmission of a particular disease.
The result of screening a single subject marker pair is a “genotype” and comprises one or more so-called alleles that are determined by the subject's DNA sequence at a marker location. Alleles are alternate forms of the DNA sequence at a genetic locus, that is, a position on a chromosome of a gene or other chromosome marker. Persons may be homozygous or heterozygous depending on the number of different alleles they possess for a given marker. Heterozygous persons have two different alleles (one from each parent) for a given marker, whereas homozygous persons inherit the same allele from both parents for a given marker.
As researchers study more complex traits and diseases, the number of genotypes required to detect linkages to such traits and diseases grows significantly. Therefore, performing accurate and high throughput genotyping becomes an important factor for genetic analysis research.
One approach for genotyping uses a large family of microsatellite markers that enable selective amplification. The typical amplification process used is the so-called “polymerase chain reaction” (PCR) technique, which involves the use of a heat stable enzyme to catalyze a synthesis of nucleic acids on pre-existing nucleic acid templates. PCR uses the polymerase enzyme and two base pair primers, one complementary to each strand, at the end of the sequence to be amplified to produce synthesized DNA strands. The synthesized DNA strands serve as templates for the same primer sequence thus permitting successive iterations of primer annealing, strand elongation, and dissociation to produce rapid and highly specific amplification of the desired sequence. The PCR technique is applied to short segments of an individual's chromosome that are known to contain a variable length tandem repeat (i.e., marker). Each possible length corresponds to a distinct allele for the particular marker. The length of the allele is measured by separating the amplified DNA segments by length in lanes on an electrophoretic gel. Because these alleles are transmitted from parent to child, they can be used to trace the inheritance characteristics of chromosomal regions.
Processing an electrophoretic gel is time consuming. To increase throughput, often several markers are multiplexed in each lane of the gel. Markers with overlapping size ranges are tagged with different colored dyes so that their alleles can be distinguished. The same dye can be used for multiple markers, as long as their size ranges do not overlap. A DNA sequencer is used to scan the gel and produce a pixelmap color-coded image in machine-readable format. The pixel information is stored as a file that can be accessed by a gene scanner to produce individual traces. Alleles are determined from these individual traces.
One conventional approach for determining alleles uses a genotyping that presents traces to human “callers” i.e., highly-trained people who visually examine the traces to determine whether or not peaks in particular traces correspond to alleles. Often two different allele callers examine traces in double-blind fashion. If both callers agree that a particular peak or pair of peaks in a trace correspond to alleles, the genotype is “called” or identified. On the other hand, if there is no agreement, the trace may be uncallable.
SUMMARY
The allele calling algorithm assumes that a typical allele morphology can be derived for each marker. This typical allele morphology is used as a target pattern during application of allele calling algorithms to the trace data. The algorithm can train itself to adjust or adapt the typical allele morphology to a given trace. The allele calling algorithm is applied to each trace in sequence. For each trace, possible alleles are tagged with a quality or reasonableness estimate and added to a global list of allele calls and associated tags.
After all possible genotypes for the trace have been examined, a set of heuristic rules are applied to the set of calls to screen out obviously bad allele calls. The heuristic rules exclude bad calls or determine whether the trace should be labeled “uncallable” due to a high degree of uncertainty.
According to the present invention, a method executed in a computer system for identifying alleles from a trace includes applying a typical shape of an allele for a marker to the trace to identify potential allele calls that match to the typical shape of the allele at the marker and assigning a quality factor to the allele calls.
According to a further aspect of the invention, a method executed in a computer system for identifying alleles from trace data includes extracting trace data from a database and preprocessing the trace data to correct for errors in the trace data. The method also includes comparing peaks in the trace data to a typical allele shape to produce potential allele calls and postprocessing the potential allele calls by applying at least one heuristic processing criterion to the potential allele calls to determine whether the potential allele calls should be an allele call.
According to a still further aspect of the invention, a computer program product residing on a computer readable medium for identifying alleles from a trace, comprises instructions for causing a computer to apply a typical shape of an allele for a marker to the trace to identify potential allele calls that match to the typical shape of the allele at the marker and assign a quality factor to the allele calls.
According to a still further aspect of the invention, a computer program product residing on a computer readable medium for identifying alleles from a trace, includes instructions for causing a computer to extract trace data from a database and preprocess the trace data to correct for errors in the trace data. The computer program product also includes instructions to cause the computer to compare peaks in the trace data to a typical allele shape to find potential allele calls, and postprocess potential allele calls by applying at least one heuristic processing criterion to the potential allele calls to determine whether the potential allele calls should be an allele call.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
REFERENCES:
patent: 5541067 (1996-07-01), Perlin
patent: 5580728 (1996-12-01), Perlin
patent: 5916747 (1999-06-01), Gilchrist et al.
Genotyper Software ABI Systems pp. 1-4, 1999.*
Stoughton et al Electrophoresis vol. 18 No. 1 pp. 1-5, 1997.*
“PCR-SSCP: A Simple and Sensitive Method for Detection of Mutations in the Genomic DNA”, K. Hayashi, PCR Methods and Applications, 1991, pp. 34-38.
“Methods for Precise Sizing, Automated Binning of Alleles, and Reduction of Error Rates in Large-Scale Genotyping Using Fluorescently Labeled Dinucleotide Markers”, S. Ghosh et al, Genome Research, 1997, pp. 165-178.
Hiller Martha J.
Martin John W.
Nicoletti Richard A.
Page Christopher R.
Parker Alexander N.
Fish & Richardson P.C.
Horlick Kenneth R.
Millennium Pharmaceuticals Inc.
Siew Jeffrey
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