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 determining methylation profiles of cell-free deoxyribonucleic acid (cfDNA) molecules, the method comprising:
(a) providing cfDNA molecules obtained from a subject;
(b) ligating adaptors and amplifying the cfDNA molecules to generate amplified cfDNA molecules;
(c) enriching the amplified cfDNA molecules using sequence capture for a panel of genes or genomic regions to generate an enriched set of amplified cfDNA molecules, wherein the panel is at least 150kb, and wherein the genes or genomic regions:
(i) are selected using information from whole genome bisulfite sequencing including from tumor samples,
(ii) comprise CpG islands which are differentially methylated regions;
(d) sequencing the enriched set of amplified cfDNA to generate sequencing data, wherein the sequencing is massively parallel sequencing that sequences at least 10 million polynucleotide molecules; and
(e) applying one or more bioinformatics processes to the sequencing data to detect methylation profiles of the cfDNA.
2. The method of claim 1 , wherein the adaptor ligation comprises single strand ligation.
3. The method of claim 1 , wherein the ligation conditions comprise the use of over 10× molar excess of adaptors.
4. The method of claim 1 , wherein the methylation profiles are used to detect a cancer in the subject, wherein the cancer is colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, or liver cancer.
5. The method of claim 1 , wherein the sequencing is massively parallel sequencing that sequences at least 100 million polynucleotide molecules.
6. The method of claim 1 , wherein the sequencing data comprises at least 1 billion base pairs.
7. The method of claim 1 , wherein one or more of the genomic regions in the panel have a size from about 0.1 kb to about 5 kb.
8. The method of claim 1 , wherein one or more of the genomic regions in the panel have a size of about 1 kb or less.
9. The method of claim 1 , further comprising determining the tissue of origin of the cfDNA.
10. The method of claim 1 , wherein the panel comprises a plurality of subpanels, including subpanels for identifying tissue of origin.
11. The method of claim 1 , wherein the cfDNA molecules have been isolated from a bodily fluid.
12. The method of claim 1 , wherein the subject has not have received a cancer treatment.
13. The method of claim 1 , wherein the subject does not have a cancer that is detectable by imaging methods.
14. The method of claim 13 , wherein the imaging method is positron emission tomography scan, magnetic resonance imaging, X-ray, computerized axial tomography scan, ultrasound, or a combination thereof.
15. The method of claim 1 , wherein the subject has experienced symptoms consistent with a diagnosis of cancer.
16. The method of claim 1 , wherein the adaptors comprise molecular barcodes.
17. The method of claim 1 , wherein a read budget is selected that identifies the total number of base reads to be allocated to the sample, wherein the sample comprises a predetermined amount of cDNA.
18. The method of claim 1 , wherein multiple cfDNA samples are analyzed which are collected from the subject over a plurality of time points.
19. The method of claim 4 , wherein the cancer is detected at a specificity of about 95% or greater.