Methods for computer processing sequence reads to detect molecular residual disease
Disclosed herein are methods for use in detection of molecular residual disease. The methods may comprise deep sequencing a panel of genomic regions in cell-free DNA molecules and computer processing sequence reads to detect variants that are indicative of molecular residual disease.
1. A method for detecting molecular residual disease in a subject, the method comprising:
(a) ligating adapters to a plurality of cell-free deoxyribonucleic acid (cfDNA) molecules from a sample of the subject to generate adapter-ligated polynucleotides;
(b) amplifying a plurality of the adapter-ligated polynucleotides to generate amplified polynucleotides;
(c) enriching a plurality of the amplified polynucleotides for a sequencing panel comprising genomic regions to generate an enriched set of polynucleotides, wherein the enriched set of polynucleotides is representative of no more than 35,000 base pairs of a human genome, wherein the sequencing panel is selected using information derived from a cancer tumor biopsy of the subject indicating at least one of the no more than 35,000 base pairs of human genome contain a tumor marker present in a greater proportion in the cancer tumor biopsy than a background population, wherein the enriching is performed using amplification-based enrichment;
(d) sequencing a plurality of the enriched set of polynucleotides at a sequence read depth of at least about 50,000 sequence reads per base to generate sequence reads;
(e) computer processing a plurality of the sequence reads at least in part by aligning a plurality of the sequence reads to a reference genome to generate aligned sequence reads; and
(f) computer processing a plurality of the aligned sequence reads to detect a variant corresponding to a tumor marker in the no more than 35,000 base pairs of human genome indicated in (c) from a plurality of the sequence reads generated in (d), thereby determining the molecular residual disease in the subject.
2. The method of claim 1 , wherein the sequencing is at a sequence read depth of at least about 100,000 sequence reads per base.
3. The method of claim 1 , further comprising comparing sequence information obtained from the plurality of amplified polynucleotides to sequence information obtained from a cohort of healthy individuals.
4. The method of claim 1 , wherein the computer processing in (f) comprises determining a plurality of base calls from the plurality of the aligned sequence reads at least in part by comparing the plurality of the aligned sequence reads with a plurality of reference sequences, wherein a base call is reported if its frequency is greater than a noise threshold as determined by the frequency in a plurality of reference sequences.
5. The method of claim 4 , wherein the reference sequences are sequences from healthy individuals.
6. The method of claim 1 , further comprising using a cohort of healthy individuals to provide a baseline frequency of a base at a position, and the baseline frequency is used in determining the plurality of base calls.
7. The method of claim 1 , further comprising comparing a frequency for a base at a position in a cohort of healthy individuals from the subject to a frequency of a base detected among sequence reads from the subject.
8. The method of claim 1 , wherein the enriched set of polynucleotides is representative of no more than 10,000 base pairs of a human genome.
9. The method of claim 1 , wherein the enriched set of polynucleotides is representative of no more than 5,000 base pairs of a human genome.
10. The method of claim 1 , wherein the cfDNA molecules are isolated from a bodily fluid sample.
11. The method of claim 10 , wherein the bodily fluid sample is a blood sample, a plasma sample, or a serum sample.
12. The method of claim 1 , wherein the subject has previously received a treatment for a cancer.
13. The method of claim 12 , wherein the cancer is selected from the group consisting of colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and liver cancer.
14. The method of claim 12 , wherein the treatment for the cancer is selected from the group consisting of a surgical treatment, a radiation treatment, a chemotherapy, a targeted cancer therapeutics, and a cancer immunotherapy.
15. The method of claim 12 , wherein the subject does not detectably exhibit any symptoms of the cancer.
16. The method of claim 1 , further comprising determining a consensus sequence from a plurality of the sequence reads to reduce errors from the amplifying in (b) or (c), or the sequencing in (d).
17. The method of claim 1 , further comprising, prior to the sequencing in (d), tagging a plurality of the enriched set of cfDNA molecules by PCR amplification using primers with barcodes.
18. The method of claim 1 , wherein the sequencing in (d) is performed within a read budget that allocates a pre-determined total number of base reads, wherein the plurality of cfDNA molecules comprises no more than a pre-determined amount of DNA.
19. The method of claim 1 , wherein multiple cfDNA samples are collected from the subject over a plurality of time points and analyzed.
20. The method of claim 1 , further comprising filtering out at least a portion of the plurality of the sequence reads or the plurality of the aligned sequence reads.