| CPC G16B 20/10 (2019.02) [C12Q 1/68 (2013.01); G16B 20/20 (2019.02); G16B 30/10 (2019.02); G16B 40/00 (2019.02)] | 17 Claims |
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1. A method for determining a presence or absence of a copy number alteration for a test subject, comprising:
obtaining circulating cell free nucleic acid from a test sample from the test subject;
ligating nucleic acid molecules of the circulating cell free nucleic acid with adapters to generate a plurality of sequence constructs, wherein each sequence construct comprises an adapter ligated to an end of a nucleic acid molecule;
generating, using a first polymerase chain reaction, library constructs for each sequence construct of the plurality of sequence constructs;
capturing a subset of the library constructs using probe oligonucleotides under hybridization conditions to enrich for one or more genomic regions of interest, wherein the probe oligonucleotides cover multiple types of genetic alterations;
generating, using a second polymerase chain reaction, enriched library constructs for each library construct of the subset of the library constructs;
sequencing the enriched library constructs to obtain sequence reads;
generating, by a computing system, an alignment computer file comprising on-target sequence reads and associated genomic positioning data, wherein the generating the alignment computer file comprises aligning the sequence reads to a reference genome and matching the aligned sequence reads to genomic sequences in a probe panel to obtain the on-target sequence reads, and wherein the genomic sequences correspond to the probe oligonucleotides;
generating, by running programming language scripts on the alignment computer file by the computing system, a probe computer file, wherein the generating the probe computer file comprises:
determining a sample position read coverage for each base in each probe oligonucleotide by quantifying the on-target sequence reads that map to each base in the genomic sequences corresponding to each probe oligonucleotide;
determining a sample read coverage for each probe oligonucleotide based on the sample position read coverage for each base in the genomic sequences corresponding to the probe oligonucleotide;
determining a reference read coverage for each probe oligonucleotide based on reference samples; and
transforming the sample read coverage for each probe oligonucleotide into a probe log 2 ratio by dividing the sample read coverage for each probe oligonucleotide by the reference read coverage for each probe oligonucleotide to obtain a ratio and logarithmically transforming the ratio according to a log 2 transformation; and
generating, by running variant caller programming language scripts on the probe computer file by the computing system, a variant call file, wherein the generating the variant call file comprises:
identifying candidate variants based on the probe log 2 ratio for each probe oligonucleotide using a variant caller method; and
determining the presence or absence of the copy number alteration in the circulating cell free nucleic acid based on the candidate variants.
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