Methods for early detection of cancer
Disclosed herein are methods for use in detection of single nucleotide variants (SNVs) or indels. The methods may comprise enriching cell-free DNA molecules for a panel of genomic regions and deep sequencing the enriched cfDNA to detect the SNVs or indels.
1. A method for preparing and sequencing nucleic acids, the method comprising:
a) providing a sample of cell-free deoxyribonucleic acid (cfDNA) molecules from a subject;
b) attaching adapters to a plurality of the cfDNA molecules by ligation to produce adapted nucleic acids;
c) amplifying a plurality of the adapted nucleic acids by polymerase chain reaction (PCR)-based amplification to provide amplified nucleic acids;
d) enriching the amplified nucleic acids for a panel of genomic regions, wherein the panel comprises one or more genomic regions from each of at most 20 genes, wherein the at most 20 genes comprises one or more genes selected from AKT1, ALK, APC, ATM, BRAF, CTNNB1, EGFR, ERBB2, ESR1, FGFR2, GATA3, GNAS, IDH1, IDH2, KIT, KRAS, MET, NRAS, PDGFRA, PIK3CA, PTEN, RB1, SMAD4, STK11, and TP53; and
e) sequencing the enriched set of amplified nucleic acids to generate sequence reads, wherein the sequencing is performed at a sequence read depth of at least 50,000 sequence reads per base, and wherein the sequencing is performed within a read budget that allocates a pre-determined total number of base reads, wherein the enriched set of amplified nucleic acids comprises no more than a pre-determined amount of DNA.
2. The method of claim 1 , wherein the adapters comprise molecular barcodes having predetermined sequences.
3. The method of claim 1 , wherein the cancer is selected from the group consisting of ovarian cancer, pancreatic cancer, breast cancer, colorectal cancer, and non-small cell lung carcinoma.
4. The method of claim 3 , wherein the cancer is non-small cell lung carcinoma (NSCLC), and the non-small cell lung carcinoma is squamous cell carcinoma or adenocarcinoma.
5. The method of claim 1 , wherein the cfDNA molecules are derived from blood, serum or plasma.
6. The method of claim 1 , wherein the subject has previously received treatment for a cancer.
7. The method of claim 3 , wherein the subject has received surgical treatment, radiation treatment, chemotherapy, targeted cancer therapeutics or a cancer immunotherapy.
8. The method of claim 1 , wherein the enriching is performed by sequence capture using oligonucleotide probes that hybridize to the sequences in the panel of genomic regions.
9. The method of claim 8 , wherein the oligonucleotide probes are 60 to 120 bases long.
10. The method of claim 1 , wherein sequencing is performed at a read depth of at least 100,000 reads per base.
11. The method of claim 1 , further comprising comparing sequence information from the cfDNA molecules to sequence information obtained from a cohort of healthy individuals, a cohort of cancer patients, or germline DNA from the subject.
12. The method of claim 1 , wherein the panel comprises at least one exon from each of the genes.
13. The method of claim 1 , wherein multiple samples comprising cfDNA are collected from the subject over a plurality of time points and enriched and sequenced.
14. The method of claim 1 , wherein the sample comprises 10 to 300 nanograms (ng) of cfDNA molecules.
15. The method of claim 1 , wherein the enriching is performed using amplification-based enrichment.
16. The method of claim 1 , wherein the panel of genomic regions is selected using information derived from a cancer tumor biopsy of the subject indicating at least one of the genomic regions contain a tumor marker present in a greater proportion in the cancer tumor biopsy than a background population.
17. The method of claim 1 , wherein the panel comprises 10 to 20 different genes.