Methods and systems for detecting genetic variants
Disclosed herein in are methods and systems for determining genetic variants (e.g., copy number variation) in a polynucleotide sample. A method for determining copy number variations includes tagging double-stranded polynucleotides with duplex tags, sequencing polynucleotides from the sample and estimating total number of polynucleotides mapping to selected genetic loci. The estimate of total number of polynucleotides can involve estimating the number of double-stranded polynucleotides in the original sample for which no sequence reads are generated. This number can be generated using the number of polynucleotides for which reads for both complementary strands are detected and reads for which only one of the two complementary strands is detected.
1. A method of monitoring a patient's disease status, wherein the method comprises:
(a) providing a sample comprising double-stranded polynucleotide molecules from the patient, wherein a double-stranded polynucleotide molecule includes first and second complementary strands;
(b) attaching adapters to a plurality of the polynucleotide molecules to generate tagged double-stranded polynucleotide molecules, wherein attaching adapters comprises ligation using more than a 10× excess of adapters as compared to the double-stranded polynucleotide molecules, wherein at least 20% of the double-stranded polynucleotide molecules are attached with adapters;
(c) amplifying a plurality of the tagged double-stranded polynucleotide molecules to produce amplified polynucleotides;
(d) sequencing at least some of amplified polynucleotides to produce a set of sequence reads;
(e) determining an amount of one or more epigenetic modifications from the set of sequence reads, wherein the set of sequence reads comprise paired reads and/or unpaired reads, wherein (i) each paired read corresponds to sequence reads generated from a first tagged strand and a second tagged complementary strand derived from a double-stranded polynucleotide molecule in said set, and (ii) each unpaired read represents a first tagged strand having no second tagged complementary strand derived from a double-stranded polynucleotide molecule represented among said sequence reads in said set of sequence reads; and
(f) repeating (a)-(e) at one or more time points and determining whether there is a difference in the amounts of epigenetic modifications between two or more time points, thereby monitoring a patient's disease status.
2. The method of claim 1 , wherein the said sample comprises double-stranded polynucleotide molecules sourced from a tumor sample.
3. The method of claim 1 , wherein said sample comprises double-stranded polynucleotide molecules sourced from cell-free nucleic acids.
4. The method of claim 1 , wherein a sample at one time point comprises double-stranded polynucleotide molecules sourced from a tumor sample and a sample at another time point is sourced from cell-free nucleic acids.
5. The method of claim 1 , wherein the adapters are double-stranded tags that are Y-shaped with a hybridized portion at one end of the tag and a non-hybridized portion at the opposite end of the tag.
6. The method of claim 1 , wherein the adapters comprise molecular barcodes.
7. The method of claim 6 , wherein the plurality of polynucleotide molecules is tagged with n different combinations of molecular barcodes, wherein n is at least 2 and no more than 100,000*z, wherein z is a mean of an expected number of duplicate molecules in the sample of double-stranded polynucleotide molecules that map to identical start and stop positions on a reference sequence.
8. The method of claim 7 , wherein n is at least 2 and no more than 1,000*z.
9. The method of claim 1 , wherein attaching adapters comprises ligation using more than an 80× excess of adapters as compared to the double-stranded polynucleotide molecules, wherein at least 40% of the double-stranded polynucleotide molecules are attached with adapters.
10. The method of claim 1 , wherein the set of sequence reads are mapped to a reference sequence.
11. The method of claim 10 , further comprising reducing and/or tracking redundancy in said set of sequence reads.
12. The method of claim 11 , wherein reducing redundancy in said set of sequence reads comprises collapsing sequence reads produced from amplified products of an original polynucleotide molecule in said sample back to said original polynucleotide molecule.
13. The method of claim 12 , wherein the collapsing back to said original polynucleotide molecule is based at least on molecular barcodes.
14. The method of claim 13 , further comprising determining a consensus sequence for said original polynucleotide molecule.
15. The method of claim 1 , wherein the determining an amount of one or more epigenetic modifications comprises identifying polynucleotide molecules at one or more genetic loci comprising the epigenetic modification.
16. The method of claim 15 , further comprising determining a quantitative measure of paired reads that map to a locus, wherein both strands of said pair comprise the epigenetic modification.
17. The method of claim 15 , further comprising determining a quantitative measure of paired molecules in which only one member of said pair bears the epigenetic modification and/or determining a quantitative measure of unpaired molecules bearing the epigenetic modification.
18. The method of claim 1 , wherein the disease is cancer.
19. The method of claim 18 , wherein the cancer is colorectal cancer or lung cancer.
20. The method of claim 1 , wherein the method further comprises selectively enriching a subset of the tagged double-stranded polynucleotide molecules, or amplification products thereof, prior to sequencing.
21. The method of claim 20 , wherein the selective enrichment is performed by amplification.
22. The method of claim 20 , wherein the selective enrichment is performed by probe-based hybridization.
23. The method of claim 1 , further comprising determining quantitative measures of (i) said paired reads and (ii) said unpaired reads that map to each of one or more genetic loci.
24. The method of claim 23 , further comprising estimating with a programmed computer processor a quantitative measure of total double-stranded polynucleotide molecules in the sample that map to each of the one or more genetic loci based on said quantitative measure of paired reads and unpaired reads mapping to each locus.
25. The method of claim 24 , further comprising detecting copy number variation in the sample by determining a normalized total quantitative measure, from the quantitative measure, at each of said one or more genetic loci and determining copy number variation based on the normalized measure.
26. A method of monitoring a patient's disease status, wherein the method comprises:
(a) providing a sample comprising double-stranded cell-free deoxyribonucleic acid (cfDNA) molecules from the patient, wherein a double-stranded polynucleotide molecule includes first and second complementary strands;
(b) attaching adapters to a plurality of the double-stranded cfDNA molecules to generate tagged double-stranded polynucleotide molecules;
(c) amplifying a plurality of the tagged double-stranded polynucleotide molecules to produce amplified polynucleotides;
(d) sequencing at least some of amplified polynucleotides to produce a set of sequence reads;
(e) determining an amount of one or more epigenetic modification from the set of sequence reads, wherein the set of sequence reads comprise paired reads and/or unpaired reads, wherein (i) each paired read corresponds to sequence reads generated from a first tagged strand and a second tagged complementary strand derived from a double-stranded cfDNA molecule in said set, and (ii) each unpaired read represents a first tagged strand having no second tagged complementary strand derived from a double-stranded cfDNA molecule represented among said sequence reads in said set of sequence reads; and
(f) repeating (a)-(e) at one or more time points and determining whether there is a difference in the amounts of epigenomic modifications between two or more time points, thereby monitoring a patient's disease status.
27. The method of claim 26 , wherein the adapters comprise molecular barcodes.
28. The method of claim 27 , wherein the plurality of cfDNA molecules is tagged with n different combinations of molecular barcodes, wherein n is at least 2 and no more than 100,000*z, wherein z is a mean of an expected number of duplicate molecules in the sample of double-stranded cfDNA molecules that map to identical start and stop positions on a reference sequence.
29. The method of claim 28 , wherein n is at least 2 and no more than 1,000*z.
30. The method of claim 28 , wherein attaching adapters comprises ligation using more than an 10× excess of adapters as compared to the double-stranded cfDNA molecules, wherein at least 20% of the double-stranded cfDNA molecules are attached with adapters.