Methods and systems for dynamic variant thresholding in a liquid biopsy assay

A method of validating a somatic sequence variant in a cancerous tissue of a test subject having a cancer condition, the method comprising:

• at a computer system having one or more processors, and memory storing one or more programs for execution by the one or more processors:
• (A) obtaining, from a first sequencing reaction, a corresponding sequence of each cell-free DNA fragment in a first plurality of cell-free DNA fragments in a liquid biopsy sample of the test subject, thereby obtaining a first plurality of sequence reads, wherein the first plurality of sequence reads comprises at least 100,000 sequence reads;
• (B) aligning each respective sequence read in the first plurality of sequence reads to a reference sequence for the species of the subject thereby identifying a candidate somatic sequence variant mapping to a respective locus in the reference sequence;
• (C) determining for the candidate somatic sequence variant, (i) a respective variant allele fragment count for the first sequencing reaction, and (ii) a respective locus fragment count for the first sequencing reaction;
• and
• (D) comparing the respective variant allele fragment count for the candidate somatic sequence variant against a dynamic variant count threshold for the respective locus in the reference sequence that the candidate variant maps to, wherein the dynamic variant count threshold is based upon at least a pre-test odds of a positive variant call for the respective locus based upon a prevalence of variants in a genomic region that includes the respective locus in a cohort of training subjects having the cancer condition, and:
• when the variant allele fragment count for the candidate somatic sequence variant satisfies the dynamic variant count threshold for the respective locus, not rejecting the presence of the candidate somatic sequence variant in the test subject, or
• when the variant allele fragment count for the candidate somatic sequence variant does not satisfy the dynamic variant count threshold for the locus, rejecting the presence of the candidate somatic sequence variant in the test subject.