METHODS FOR EARLY DETECTION OF CANCER
Disclosed herein are methods, compositions, and devices for use in the early detection of cancer. The methods include preparing cell-free nucleic acid molecules from a subject for sequencing, sequencing a panel of regions in the cell-free nucleic acid molecules, and detecting one or more markers that are indicative of a cancer.
1 . A method for detecting molecular residual disease in a subject, the method comprising:
(a) obtaining a plurality of polynucleotides which are or derive from cell-free deoxyribonucleic acid (cfDNA) molecules of the subject;
(b) enriching a plurality of the polynucleotides for a sequencing panel of genomic regions to generate an enriched set of polynucleotides, wherein the sequencing panel is selected using information derived from a cancer tumor biopsy of the subject, and wherein the enriching is performed using capture probes specific for genomic regions comprising single-nucleotide variants;
(c) sequencing a plurality of the enriched set of polynucleotides at a sequence read depth of at least about five thousand sequence reads per base to generate sequence reads;
(d) 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;
(e) computer processing a plurality of the aligned sequence reads to detect a variant corresponding to at least one of the single-nucleotide variants, thereby determining the molecular residual disease in the subject.
2 . The method of claim 1 , wherein the cfDNA molecules are uniquely tagged with respect to one another.
3 . The method of claim 1 , the method further comprising amplifying the cfDNA prior to sequencing, and determining a consensus sequence from sequence reads obtained from the sequencing to reduce errors from amplification or sequencing.
4 . The method of claim 3 , wherein the determining the consensus sequence is performed on a molecule-by-molecule basis.
5 . The method of claim 3 , wherein the determining the consensus sequence is performed on a base by base basis.
6 . The method of claim 3 , wherein the determining the consensus sequence is performed using molecular barcodes that tag individual cfDNA molecules derived from the subject.
7 . The method of claim 1 , further comprising comparing sequence information obtained from the plurality of polynucleotides to sequence information obtained from a cohort of healthy individuals.
8 . The method of claim 1 , wherein the enriched set of polynucleotides is representative of more than 100,000 bases of the human genome.
9 . The method of claim 1 , wherein the cfDNA molecules are isolated from a bodily fluid sample of the subject.
10 . The method of claim 9 , wherein the bodily fluid sample is a blood sample, a plasma sample, or a serum sample.
11 . The method of claim 1 , wherein the subject has previously been diagnosed with cancer.
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 , wherein the sequencing 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.
17 . The method of claim 1 , wherein multiple cfDNA samples are collected from the subject over a plurality of time points and analyzed.
18 . The method of claim 1 , wherein the sequencing panel is selected to achieve a sensitivity of at least 85% for a cancer selected from the group consisting of colorectal cancer, ovarian cancer, lung cancer, and pancreatic cancer.
19 . The method of claim 1 , wherein the cfDNA molecules are tagged with duplex tags which differentially label the complementary strands of the cfDNA.
20 . The method of claim 19 , wherein the tags are Y-shaped adaptors comprising barcodes that are between 2 and 32 nucleotides in length.