Methods and systems for detecting genetic variants
Inventors
Talasaz, AmirAli • Eltoukhy, Helmy • MORTIMER, Stefanie Ann Ward
Assignees
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Publication Number
US-12286672-B2
Publication Date
2025-04-29
Expiration Date
Abstract
Disclosed herein in are methods and systems for determining genetic variants (e.g., copy number variation) in a polynucleotide sample. A method for determining copy number variations includes tagging double-stranded polynucleotides with duplex tags, sequencing polynucleotides from the sample and estimating total number of polynucleotides mapping to selected genetic loci. The estimate of total number of polynucleotides can involve estimating the number of double-stranded polynucleotides in the original sample for which no sequence reads are generated. This number can be generated using the number of polynucleotides for which reads for both complementary strands are detected and reads for which only one of the two complementary strands is detected.
Core Innovation
The invention relates to treating a subject having a cancer associated with one or more genetic variants by selecting the subject using cell-free deoxyribonucleic acid (cfDNA) analysis and then treating the subject based on the determining results. A sample of cfDNA molecules from the subject is used to ligate adapters to produce tagged parent polynucleotides, and the tagged polynucleotides are amplified to produce tagged progeny polynucleotides. The tagged progeny polynucleotides are enriched with a sequencing panel to produce enriched polynucleotides, and one or more genomic regions from each gene of the sequencing panel are sequenced to generate sequence reads.
The invention uses a sequencing panel having oligonucleotide probes comprising genomic regions from a plurality of genes associated with cancer. Based on the plurality of sequence reads, the method determines the presence of one or more genetic variants from the one or more genomic regions. The subject is then treated with a therapy based on determining the presence of the one or more genetic variants.
The disclosed approaches also include molecular identifier and read-processing strategies, including tagging cfDNA molecules and generating consensus sequences, and calling variants using grouped read families and normalization against reference loci. The described workflow includes comparing quantities at loci and/or reference loci, and polyploidy inference using ratio-based approaches, including references such as HG19 (GRCh37) and GRCh38. The document further describes supporting system and software components that group reads into families, generate consensus sequences, and call genetic variants including CNV and targeted gene variants, including limits of detection (LOD) determination for targeted genes.
Claims Coverage
Independent claim coverage is explicitly provided for one treating method using cfDNA analysis with adapter tagging, panel enrichment, sequencing, variant determination, and therapy.
CfDNA adapter tagging, amplification, and panel enrichment for variant-guided therapy
Provide a sample of cell-free deoxyribonucleic acid (cfDNA) molecules from a subject having cancer, ligate adapters to produce tagged parent polynucleotides, amplify to produce tagged progeny polynucleotides, enrich the tagged progeny polynucleotides with a sequencing panel comprising oligonucleotide probes for genomic regions from a plurality of genes, sequence genomic regions to generate sequence reads, determine presence of one or more genetic variants from the genomic regions, and treat the subject with therapy based on determining the presence of the one or more genetic variants.
The explicitly described independent claim covers selecting and treating a cancer subject by detecting genetic variants from cfDNA using adapter ligation and tagging, amplification, multi-gene sequencing panel enrichment, sequencing, variant determination, and therapy selection based on the detected variants.
Stated Advantages
Improves base calling.
Enables more accurate locus quantification for variant detection and copy number variation (CNV).
Supports more accurate detection of unseen molecules by inferring unseen molecule counts.
Enables higher specificity and detection of rare DNA/cancer-associated variants.
Documented Applications
Detecting rare DNA/cancer-associated genetic variants at high specificity.
Monitoring disease status using cfDNA analysis and therapy selection based on detected variants.
Pooled test and control workflows to reduce bias.
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