METHODS AND SYSTEMS FOR DETECTING GENETIC VARIANTS

• US 20160046986A1
• Filed: 09/22/2015
• Published: 02/18/2016
• Est. Priority Date: 12/28/2013
• Status: Active Grant

• Current Assignee: Guardant Health Inc.
• Original Assignee: Guardant Health Inc.
• Inventors: Eltoukhy, Helmy, Talasaz, AmirAli
• Granted Patent: US 9,920,366 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment

Abstract

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.

Claims

1. A method for estimating a total number of double-stranded deoxyribonucleic acid (DNA) molecules in a sample, comprising:

(a) determining a quantitative measure of individual DNA molecules for which both strands are detected;

(b) determining a quantitative measure of individual DNA molecules for which only one strand is detected;

(c) using said quantitative measures determined in (a) and (b) to estimate a total number of double-stranded DNA molecules in the sample, wherein said total number comprises individual DNA molecules for which neither DNA strand is detected.

2. The method of claim 1, further comprising inferring, from said quantitative measures determined in (a) and (b), a quantitative measure of individual DNA molecules for which neither strand is detected.

3. The method of claim 1, further comprising determining a normalized quantitative measure of one or more genetic loci to determine copy number variation in said sample.

4. The method of claim 1, wherein said sample comprises double-stranded polynucleotide molecules sourced substantially from cell-free nucleic acids.

5. The method of claim 1, wherein determining said quantitative measure of individual DNA molecules comprises tagging said DNA molecules with a set of duplex tags, wherein each duplex tag differently tags complementary strands of a double-stranded DNA molecule in said sample to provide tagged strands.

6. The method of claim 5, further comprising sequencing at least some of said tagged strands to produce a set of sequence reads.

7. The method of claim 6, further comprising sorting sequence reads into paired reads and unpaired reads, wherein (i) each paired read corresponds to sequence reads generated from a first tagged strand and a second differently 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 differently tagged complementary strand derived from a double-stranded polynucleotide molecule represented among said sequence reads in said set of sequence reads.

8. The method of claim 7, 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 to determine a quantitative measure of total double-stranded DNA molecules in said sample that map to each of said one or more genetic loci based on said quantitative measure of paired reads and unpaired reads mapping to each locus.

9. A method, comprising:

(a) providing a sample comprising a set of double-stranded polynucleotide molecules, each double-stranded polynucleotide molecule including first and second complementary strands;

(b) tagging said double-stranded polynucleotide molecules with a set of duplex tags, wherein each duplex tag differently tags said first and second complementary strands of a double-stranded polynucleotide molecule in said set;

(c) sequencing at least some of said tagged strands to produce a set of sequence reads;

(d) reducing and/or tracking redundancy in said set of sequence reads;

(e) sorting sequence reads into paired reads and unpaired reads, wherein (i) each paired read corresponds to sequence reads generated from a first tagged strand and a second differently 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 differently tag complementary strand derived from a double-stranded polynucleotide molecule represented among said sequence reads in said set of sequence reads;

(f) determining quantitative measures of at least two of (i) said paired reads, (ii) said unpaired reads that map to each of one or more genetic loci, (iii) read depth of said paired reads and (iv) read depth of said unpaired reads; and

(g) estimating with a programmed computer processor a quantitative measure of total double-stranded polynucleotide molecules in said set that map to each of said one or more genetic loci based on said quantitative measures of said at least two of (i) said paired reads, (ii) said unpaired reads mapping to each locus, (iii) said read depth of said paired reads and (iv) said read depth of said unpaired reads.

10. The method of claim 9, further comprising detecting copy number variation in said sample by determining a normalized total quantitative measure determined in (g) at each of said one or more genetic loci and determining copy number variation based on the normalized measure.

11. The method of claim 9, wherein said sample comprises double-stranded polynucleotide molecules sourced substantially from cell-free nucleic acids.

12. The method of claim 9, wherein said duplex tags are not sequencing adaptors.

13. The method of claim 9, wherein (f) comprises determining quantitative measures of said paired reads and said unpaired reads, and wherein in (g), said quantitative measure of total double-stranded polynucleotide molecules in said set that map to each of said one or more genetic loci is determined based on said quantitative measures of said paired reads and said unpaired reads.

14. The method of claim 9, 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.

15. The method of claim 14, further comprising determining a consensus sequence for said original polynucleotide molecule.

16. The method of claim 15, further comprising identifying polynucleotide molecules at one or more genetic loci comprising a sequence variant.

17. 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 a sequence variant.

18. The method of claim 15, further comprising determining a quantitative measure of paired molecules in which only one member of said pair bears a sequence variant and/or determining a quantitative measure of unpaired molecules bearing a sequence variant.

19. A method for detecting copy number variation in deoxyribonucleic acid (DNA) molecules in a biological sample of a subject, comprising:

(a) attaching adapters to ends of fragments generated from said DNA molecules in said biological sample of said subject, wherein said adapters tag a 5′

end of a strand of an individual fragment among said fragments with a first tag and a 3′

end of a complementary strand of said individual fragment with a second tag, thereby providing tagged fragment molecules;

(b) sequencing at least a portion of each of said tagged fragment molecules to provide a plurality of sequencing reads;

(c) mapping said plurality of sequencing reads to a first genetic locus and at least one second genetic locus in a reference genome, wherein said first tag and said second tag are indicative of which strand of said tagged fragment molecules each of said plurality of sequencing reads is derived;

(d) using a programmed computer to determine a first total number of tagged fragment molecules for said first genetic locus and a second total number of tagged fragment molecules for said at least one second genetic locus, wherein each of said first total number and second total number is based on (i) a number of tagged fragments for which sequencing reads from both strands of said tagged fragment molecules are detected and (ii) a number of tagged fragment for which sequencing reads from only one strand of said tagged fragment molecules are detected, and wherein said first total number and said second total number comprises tagged fragment molecules for which neither strand was sequenced; and

(e) comparing said first total number of tagged fragment molecules to said second total number of tagged fragment molecules to determine copy number variation in said DNA molecules.

20. The method of claim 19, further comprising detecting sequencing errors by comparing said sequencing reads derived from different strands of said tagged fragment molecules.

21. The method of claim 19, further comprising amplifying said tagged fragment molecules prior to said sequencing and, subsequent to (b), collapsing said sequencing reads.

22. The method of claim 21, further comprising detecting amplification errors by comparing said sequencing reads derived from different strands of said tagged fragment molecules.

23. The method of claim 19, wherein said copy number variation is determined if said first total number is different from said second total number.

24. The method of claim 19, wherein said biological sample is a cell-free biological sample.

25. The method of claim 19, wherein said adapters comprise molecular barcodes.

26. The method of claim 25, wherein said molecular barcodes are non-unique.

27. The method of claim 26, wherein said molecular barcodes are non-unique and at least 50% of said tagged fragment molecules comprise a given molecular barcode that is shared by at least one other tagged fragment molecule.

28. The method of claim 25, wherein said molecular barcodes are unique.

29. The method of claim 25, further comprising amplifying said tagged fragment molecules prior to said sequencing and, subsequent to (b), collapsing said sequencing reads based at least in part on said molecular barcodes.

30. The method of claim 19, wherein said copy-number variation in said DNA molecules is indicative of copy-number variation within genomes of cells in a tumor.