LOW-COVERAGE, GENOME-WIDE IDENTIFICATION OF MINORITY cfDNA CONTRIBUTORS
In some aspects, the present disclosure provides a method for analyzing cell free DNA (cfDNA). The method can comprise obtaining a biological sample derived from a subject, wherein the biological sample comprises cfDNA. The method can comprise enriching a proportion of cfDNA within the biological sample. The method can comprise sequencing the cfDNA enriched biological sample using low-coverage, genome-wide nucleic acid sequencing. The method can comprise identifying a plurality of minority components present in the sequenced cfDNA enriched biological sample. The method can comprise assigning a designation that represents a low-confidence estimate of minor variant frequency to individual identified minority components present in the sequenced cfDNA enriched biological sample. The method can comprise averaging a plurality of low-confidence estimates of minor variant frequency across a plurality of sequenced genomic loci to produce an estimation of minority component frequency in the cfDNA enriched biological sample.
1 . A method for analyzing cell free DNA (cfDNA), the method comprising:
a) obtaining a biological sample derived from a subject, wherein the biological sample comprises cfDNA;
b) enriching a proportion of cfDNA within the biological sample;
c) sequencing the cfDNA enriched biological sample using low-coverage, genome-wide nucleic acid sequencing;
d) identifying a plurality of minority components present in the sequenced cfDNA enriched biological sample;
e) assigning a designation that represents a low-confidence estimate of minor variant frequency to individual identified minority components present in the sequenced cfDNA enriched biological sample; and
f) averaging a plurality of low-confidence estimates of minor variant frequency across a plurality of sequenced genomic loci to produce an estimation of minority component frequency in the cfDNA enriched biological sample.
2 . The method of claim 1 , wherein the designation in e) is a binary classifier for the individual identified minority components to distinguish a plurality of sequenced genomic loci from sequenced genomic loci not identified as having a minor variant in the sequenced cfDNA enriched biological sample.
3 . The method of claim 1 , wherein the identifying in d) comprises:
i. aligning raw sequence data generated with low-coverage whole genome sequencing (lcWGS) in c) to a reference sequence;
ii. marking duplicate reads of sequenced fragments;
iii. conducting pre-processing of BAM files generated following lcWGS by base quality score recalibration (BQSR);
iv. performing local realignment of sequences from pre-processed BAM files to produce analysis-ready BAM files; and
v. performing variant calling on analysis-ready BAM files to identify the minority components.
4 . The method of claim 1 , wherein a reference sample comprising genomic DNA derived from the subject is analyzed to distinguish somatic genotypes present in the subject from identified minority components in the cfDNA enriched biological sample.
5 . The method of claim 4 , wherein the somatic genotypes are identified through high confidence genotyping.
6 . (canceled)
7 . The method of claim 1 , wherein the sites sequenced with low-coverage, genome-wide nucleic acid sequencing are agnostic to pre-defined genomic loci.
8 . The method of claim 1 , wherein the estimation of minority component frequency in the cfDNA enriched biological sample is a quantitative detection of a minority component present in the cfDNA of the biological sample.
9 .- 15 . (canceled)
16 . The method of claim 1 , wherein the individual identified minority components in cfDNA in e) comprise alternate heterozygous alleles or alternate homozygous alleles when compared to the alleles of genomic DNA from the subject.
17 . (canceled)
18 . The method of claim 1 , wherein the variants detected comprise single-nucleotide polymorphisms (SNPs), small insertions or deletions (INDELs), variable number of tandem repeats (VNTR), simple sequence repeats (SSR), simple tandem repeats (STR), or any combination thereof.
19 .- 22 . (canceled)
23 . The method of claim 16 , wherein the alternate heterozygous alleles or alternate homozygous alleles are derived from cfDNA from pre-malignant or malignant cells of the subject, derived from cfDNA from one or more infectious agents residing within the subject, or derived from a donor subject.
24 . (canceled)
25 . (canceled)
26 . The method of claim 23 , wherein the donor subject is an embryo or a fetus.
27 . (canceled)
28 . The method of claim 23 , wherein the donor subject has provided a tissue or an organ transplant into the subject which serves as a host.
29 . The method of claim 1 , wherein the estimation of minority component frequency in the cfDNA enriched biological sample in f) is used for transplantation monitoring, or for oncology detection or oncology monitoring.
30 .- 42 . (canceled)
43 . The method of claim 23 , comprising monitoring progression of an infectious disease.
44 .- 46 . (canceled)
47 . The method of claim 23 , wherein a development or progression of pregnancy complications is monitored.
48 . The method of claim 1 , further comprising analyzing fragment patterning in the cfDNA enriched biological sample.
49 . The method of claim 1 , further comprising imputing missing SNP genotypes in the sequenced cfDNA enriched biological sample.
50 . The method of claim 1 , further comprising calculating and evaluating regional linkage disequilibrium ratios between variant alleles detected to enhance the calculated estimation of minority component frequency in the cfDNA enriched biological sample.
51 . The method of claim 1 , further comprising individual subject level tuning comprising longitudinal sampling of biological samples obtained from the subject to screen for change in minor cfDNA components over time.
52 . The method of claim 1 , wherein the low-coverage, genome-wide nucleic acid sequencing is unbiased sequencing.
53 . (canceled)
54 . (canceled)