Methods and systems for detecting genetic variants
Inventors
Talasaz, AmirAli • Eltoukhy, Helmy • MORTIMER, Stefanie Ann Ward
Assignees
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Publication Number
US-12319961-B1
Publication Date
2025-06-03
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 monitoring a patient's disease status by analyzing double-stranded polynucleotide molecules from the patient and determining whether there is a difference in amounts of SNVs, level of SNVs, or copy numbers between two or more time points. The method provides a sample containing double-stranded polynucleotide molecules, attaches adapters to generate tagged double-stranded polynucleotide molecules, amplifies the tagged molecules, and sequences them to obtain a set of sequence reads from which an amount of one or more SNVs or copy numbers of a plurality of genes is determined.
The method characterizes the sequence reads as paired reads and/or unpaired reads, where each paired read corresponds to sequence reads generated from a first tagged strand and a second tagged complementary strand derived from a double-stranded molecule, and each unpaired read represents a first tagged strand having no second tagged complementary strand represented among the reads. By treating the paired/unpaired read structures explicitly during SNV and copy number determination, the method supports monitoring across time points for changes in SNV amounts/levels and gene copy numbers.
In one aspect, the approach is applied to double-stranded cell-free deoxyribonucleic acid (cfDNA) molecules from the patient and similarly includes adapter attachment, amplification, sequencing, and determining amounts of SNVs or copy numbers of genes from the paired/unpaired reads, followed by repeating at one or more time points to determine differences for disease monitoring. In further refinements, adapters comprise molecular barcodes with quantified barcode-combination relationships within the monitoring workflow.
Claims Coverage
The document includes two independent claims that cover monitoring a patient's disease status by sequencing-tagging-amplifying double-stranded polynucleotide or double-stranded cfDNA molecules, determining amounts of SNVs and/or gene copy numbers from paired and/or unpaired reads, and repeating the workflow at one or more time points to determine differences. Across the independent claims, the inventive features focus on paired/unpaired read definitions for variant and copy number quantification and adapter attachment and amplification/sequencing workflow.
Paired and unpaired read-based SNV and copy number determination across time points
Sequencing amplified tagged molecules to produce a set of sequence reads, determining an amount of one or more SNVs or copy numbers of a plurality of genes from the set of sequence reads, where the set comprises paired reads and/or unpaired reads defined by whether complementary tagged strands from the same double-stranded molecule are represented, and repeating at one or more time points to determine differences in SNV amounts/levels or copy numbers for monitoring a patient's disease status.
Monitoring disease status using double-stranded polynucleotide sampling with adapter attachment, amplification, and sequencing
Providing a sample comprising double-stranded polynucleotide molecules from the patient, attaching adapters to a plurality of the polynucleotide molecules to generate tagged double-stranded polynucleotide molecules, amplifying the tagged molecules to produce amplified polynucleotides, sequencing amplified polynucleotides to produce sequence reads, determining amounts of SNVs or copy numbers of a plurality of genes from the sequence reads, and repeating at one or more time points to determine whether there is a difference thereby monitoring disease status.
Monitoring disease status using double-stranded cfDNA sampling with paired and unpaired read interpretation
Providing a sample comprising double-stranded cell-free deoxyribonucleic acid (cfDNA) molecules from the patient, attaching adapters to generate tagged double-stranded polynucleotide molecules, amplifying tagged molecules, sequencing to produce sequence reads, determining amounts of SNVs or copy numbers of a plurality of genes from paired and/or unpaired reads defined by tagged complementary strand representation, and repeating at one or more time points to determine differences thereby monitoring disease status.
Overall, the independent claims jointly define a disease-monitoring method that hinges on adapter tagging of double-stranded nucleic acids, sequencing, and determining SNV amounts and/or gene copy numbers from explicitly defined paired and unpaired reads, with repetition across time points to detect differences. The cfDNA-focused independent claim mirrors the polynucleotide-based structure and grounds the paired/unpaired read definitions in tagged cfDNA complementary strands.
Stated Advantages
Detecting whether there is a difference in the amounts of SNVs, level of SNVs, or copy numbers between two or more time points for monitoring a patient's disease status.
Documented Applications
Patient disease monitoring over time using differences in SNV amounts/levels or copy numbers.
Inference of tumor heterogeneity.
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