Methods and materials for assessing allelic imbalance
Inventors
Gutin, Alexander • Timms, Kirsten • Lanchbury, Jerry
Assignees
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Publication Number
US-9574229-B2
Publication Date
2017-02-21
Expiration Date
Abstract
Methods and systems for detecting allelic imbalance using nucleic acid sequencing are provided.
Core Innovation
The invention relates to an in vitro method for detecting allelic imbalance at a plurality of single nucleotide polymorphism loci in a formalin-fixed paraffin-embedded sample comprising at least one tumor cell obtained from a patient. Genomic DNA of the at least one tumor cell is extracted to produce at least one solution, and the solution is enriched for test DNA molecules containing loci from a plurality of single nucleotide polymorphism loci. The plurality comprises at least 1,000 single nucleotide polymorphism loci, with at least one locus located on average every 5 Mb within each chromosome analyzed.
The enriched test DNA molecules are sequenced to detect the genotype of each locus in the plurality of single nucleotide polymorphism loci. Allelic imbalance is determined based on whether each locus in the plurality has allelic imbalance, including by detecting whether each locus is homozygous. The method further determines the lengths of regions of allelic imbalance in the genome of the tumor cell by detecting stretches of single nucleotide polymorphism loci in which allelic imbalance is detected.
The invention supports improved data quality on formalin-fixed paraffin-embedded tumor samples and describes sequencing-based genotype determination in place of SNP microarrays. It describes targeted enrichment and large-scale SNP panel locus-selection principles for defining and distributing the single nucleotide polymorphism loci across chromosomes, and it characterizes genomic stretches of allelic imbalance for locus-based calling and region-length determination.
Claims Coverage
Independent claims clm-00001 and clm-00022 cover sequencing-based detection of allelic imbalance across a plurality of SNP loci from formalin-fixed paraffin-embedded tumor samples, with inventive features focused on SNP-locus enrichment, sequencing-based genotyping, and calling and characterizing allelic-imbalance loci and stretches.
Sequencing-based detection of allelic imbalance from FFPE tumor DNA
An in vitro method for detecting allelic imbalance at a plurality of single nucleotide polymorphism loci, comprising extracting DNA from a formalin-fixed paraffin-embedded sample comprising at least one tumor cell obtained from a patient to produce at least one solution comprising genomic DNA of the at least one tumor cell; enriching the at least one solution for test DNA molecules each comprising at least one locus from the plurality of single nucleotide polymorphism loci, wherein the plurality comprises at least 1,000 single nucleotide polymorphism loci and wherein at least one single nucleotide polymorphism locus is located on average every 5Mb within each chromosome analyzed; performing a DNA sequencing reaction on the at least one enriched solution to detect the genotype of each locus in the plurality; and determining the lengths of a plurality of regions of allelic imbalance by detecting stretches of single nucleotide polymorphism loci in which allelic imbalance is detected.
FFPE sequencing to call allelic imbalance per SNP locus
An in vitro method for detecting allelic imbalance at a plurality of single nucleotide polymorphism loci, comprising providing a formalin-fixed paraffin-embedded sample comprising at least one tumor cell obtained from a patient; enriching the sample for test DNA molecules each comprising at least one locus from the plurality of single nucleotide polymorphism loci, wherein the plurality comprises at least 1,000 single nucleotide polymorphism loci and wherein there is at least one single nucleotide polymorphism locus located on average every 5 Mb within each chromosome; and performing at least one sequencing reaction on the test DNA molecules to detect the genotype of each locus in the plurality of single nucleotide polymorphism loci to determine whether each locus has allelic imbalance.
Across the independent claims, the core claim coverage is the combination of FFPE tumor DNA extraction, enrichment of test DNA molecules for a large distributed set of SNP loci, sequencing to genotype each locus, and determination of allelic imbalance either by forming stretches or regions of loci with allelic imbalance or by determining allelic imbalance at each locus.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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