Methods for detecting and suppressing alignment errors caused by fusion events
Inventors
ARTIERI, Carlo • Sikora, Marcin
Assignees
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Publication Number
US-12322476-B2
Publication Date
2025-06-03
Expiration Date
Abstract
Methods for producing a filtered sequencing data set by identifying one or more split sequence reads in a set of test sequence reads obtained from cell-free nucleic acid (cfNA) in a biological sample, wherein each split sequence read comprises at least one breakpoint; and, suppressing, in the set of test sequence reads, (i) at least a portion of one or more of the split sequence reads and/or at least a portion of one or more of the test sequence reads that comprise at least one sequence variant within a selected number of nucleotides from a given breakpoint, (ii) one or more base calls of the split sequence reads and/or one or more base calls of the test sequence reads that comprise at least one sequence variant within a selected number of nucleotides from a given breakpoint, thereby producing the filtered sequence information data set.
Core Innovation
The invention relates to treating a subject having lung cancer by using cell-free nucleic acid molecules from the subject and sequencing to generate sequence information comprising genetic sequence reads. The genetic sequence reads are aligned to a reference sequence that is a human genome to produce aligned sequence reads, and from the aligned sequence reads a set of gene fusion reads is identified that comprise an intragenic fusion breakpoint.
An alignment error in a subset of one or more of the gene fusion reads is detected by identifying a potential genetic variant compared to the reference sequence. The potential genetic variant is identified as being up to 20 nucleotides adjacent to the intragenic fusion breakpoint and is further constrained by a mutant allele fraction that is less than or equal to a mutant allele fraction of the intragenic fusion breakpoint in the biological sample.
The detected alignment error is filtered out from the subset of the one or more gene fusion reads to produce filtered sequence reads, thereby removing the alignment error and decreasing detection of false positive variants. Filtered sequence reads that include a single nucleotide variant (SNV) or an insertion or deletion (indel) are then determined as compared to the reference sequence indicating that the SNV or indel is present in the biological sample of the subject.
Claims Coverage
The partial document includes one independent claim. The independent claim covers treating a subject with lung cancer through cell-free nucleic acid sequencing, reference alignment to a human genome, identification of gene fusion reads with an intragenic fusion breakpoint, detection of alignment errors using a nucleotide window and mutant allele fraction constraint, filtering those errors, variant determination, and administering a selected immunotherapeutic agent based on the detected variants.
Gene fusion reads with an intragenic fusion breakpoint identification
Identifying a set of gene fusion reads that comprise an intragenic fusion breakpoint from the aligned sequence reads, where the reference sequence is a human genome.
Alignment error detection near the intragenic fusion breakpoint using mutant allele fraction constraint
Detecting an alignment error in a subset of one or more of the gene fusion reads by identifying a potential genetic variant compared to the reference sequence that is up to 20 nucleotides adjacent to the intragenic fusion breakpoint and has a mutant allele fraction less than or equal to the mutant allele fraction of the intragenic fusion breakpoint in the biological sample.
Filtering out alignment errors to decrease false positive variant detection
Filtering out the alignment error in the subset of the one or more gene fusion reads to produce filtered sequence reads, thereby removing the alignment error and decreasing detection of false positive variants.
Variant determination of SNVs or indels in filtered sequence reads versus the reference
Determining filtered sequence reads that include a single nucleotide variant (SNV) or an insertion or deletion (indel) as compared to the reference sequence indicating that the SNV or indel is present in the biological sample of the subject.
Immunotherapeutic administration selected from checkpoint inhibitor options based on detected SNVs/indels
Administering an immunotherapeutic agent to the subject based on the determining of one or more SNVs or indels to treat the lung cancer, wherein the immunotherapeutic agent is selected from pembrolizumab, nivolumab, ipilimumab, atezolizumab, avelumab, and durvalumab.
The inventive core is a workflow that uses intragenic fusion breakpoint-associated reads to detect and filter alignment errors within a nucleotide window constrained by mutant allele fraction, then calls SNVs or indels from filtered reads and uses those variants to select and administer an immunotherapeutic agent for treating lung cancer.
Stated Advantages
Decreasing detection of false positive variants by removing alignment errors from gene fusion read subsets.
More accurate true variant calling is supported by producing filtered sequence reads after suppressing alignment errors associated with gene fusion artifacts.
Documented Applications
Treating a subject having lung cancer by using cfNA/cfDNA sequencing, detecting and filtering alignment errors near an intragenic fusion breakpoint, determining SNVs/indels, and administering an immunotherapeutic agent selected from pembrolizumab, nivolumab, ipilimumab, atezolizumab, avelumab, and durvalumab based on the detected variants.
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