Sequencing methods

• US 10,395,758 B2
• Filed: 08/27/2014
• Issued: 08/27/2019
• Est. Priority Date: 08/30/2013
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Schnall-Levin, Michael
• Primary Examiner(s): Woitach, Joseph
• Application Number:
  US14/470,746
• Publication Number:
  US 20150066385A1
• Time in Patent Office: 1,826 Days
• Field of Search: None
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

The invention described herein solves challenges in providing a proficient, rapid and meaningful analysis of sequencing data. Methods and computer program products of the invention allow for a system to receive, analyze, and display sequencing data in real-time. The invention provides solutions to several difficulties encountered in assembling short sequencing-reads, and by doing so the invention improves the worth and significance of sequencing data.

Claims

1. A method of processing a plurality of nucleic acid molecules derived from a biological sample of a subject comprising:

(a) obtaining said plurality of nucleic acid molecules derived from the biological sample of said subject;

(b) using a sequencer to sequence said plurality of nucleic acid molecules to generate a set of partial sequencing reads comprising a first partial sequencing read having a first barcode sequence;

(c) while said sequencer is sequencing said plurality of nucleic acid molecules in (b), streaming said set of partial sequencing reads over a network to a sequencing analysis system remotely located with respect to said sequencer; and

(d) while said sequencer is sequencing said plurality of nucleic acid molecules in (b), using said sequencing analysis system to (i) assign said first partial sequencing read to a given group of a plurality of groups based on said first barcode sequence, (ii) correct a sequencing error identified based on a frequency of a subsequence occurring among partial sequencing reads of said given group, and (iii) assemble said first sequencing read with other reads of said set of partial sequencing reads.

2. The method of claim 1, wherein said set of partial sequencing reads comprises a second partial sequencing read having a second barcode sequence.

3. The method of claim 2, further comprising processing said first partial sequencing read and said second partial sequencing read to determine whether said first partial sequencing read and said second partial sequencing read share a common subsequence.

4. The method of claim 2, further comprising determining whether said first partial sequencing read and said second partial sequencing read correspond to a common biomolecule.

5. The method of claim 2, further comprising aligning said first partial sequencing read and said second partial sequencing read.

6. The method of claim 2, further comprising determining whether said first partial sequencing read and said second partial sequencing read share a common barcode sequence.

7. The method of claim 1, further comprising processing said first partial sequencing read with a reference sequence.

8. The method of claim 1, further comprising processing said first partial sequencing read with a reference sequence to identify a mutation in said biological sample.

9. The method of claim 3, further comprising identifying a single nucleotide polymorphism in said biological sample based on said processing.

10. The method of claim 3, further comprising identifying a translocation in said biological sample based on said processing.

11. The method of claim 1, wherein said sequencing analysis system determines a candidate sequence, wherein said candidate sequence incorporates a portion of said first partial sequencing read.

12. The method of claim 11, wherein said sequencing analysis system receives a number of partial sequencing reads, and wherein determining said candidate sequence is performed at a rate that increases as said number of partial sequencing reads increases.

13. The method of claim 1, wherein said sequencing analysis system determines to which haplotype in a pair of haplotypes said first partial sequencing read corresponds.

14. The method of claim 1, wherein said nucleic acid molecules are associated with a plurality of distinct barcodes.

15. The method of claim 1, wherein said set of partial sequencing reads comprises deoxyribonucleic acid (DNA) sequencing data.

16. The method of claim 1, wherein said set of partial sequencing reads comprises ribonucleic acid (RNA) sequencing data.

17. The method of claim 1, wherein said set of partial sequencing reads comprises genomic sequencing data.

18. A method of processing a plurality of nucleic acid molecules derived from a biological sample of a subject, comprising:

(a) obtaining said plurality of nucleic acid molecules derived from said biological sample of said subject;

(b) using a sequencer to sequence said plurality of nucleic acid molecules to generate a set of partial sequencing reads comprising a partial sequencing read having a barcode sequence; and

(c) while said sequencer is sequencing said plurality of nucleic acid molecules in (b), streaming said set of partial sequencing reads over a network to a sequencing analysis system remotely located with respect to said sequencer, wherein said sequencing analysis system comprises a database that stores said set of partial sequencing reads, and wherein said set of partial sequencing reads is analyzed in real-time by said remote sequencing analysis system while said sequencer is sequencing said plurality of nucleic acid molecules.

19. The method of claim 18, wherein said set of partial sequencing reads comprises an additional partial sequencing read having an additional barcode sequence.

20. The method of claim 19, wherein said partial sequencing read and said additional partial sequencing read share a common subsequence.

21. The method of claim 19, wherein said partial sequencing read and said additional partial sequencing read are from a common biomolecule.

22. The method of claim 19, wherein (c) comprises aligning said partial sequencing read and said additional partial sequencing read.

23. The method of claim 19, wherein said partial sequencing read and said additional partial sequencing read share a common barcode sequence.

30. The method of claim 18, further comprising processing said partial sequencing read with a reference sequence.

31. The method of claim 18, further comprising processing said partial sequencing read with a reference sequence to identify a mutation in said biological sample.

32. The method of claim 18, wherein said sequencing analysis system determines a candidate sequence, wherein said candidate sequence incorporates a portion of said partial sequencing read.

33. The method of claim 32, wherein said sequencing analysis system receives a number of partial sequencing reads, and wherein determining said candidate sequence is performed at a rate that increases as said number of partial sequencing reads increases.

34. The method of claim 18, wherein said sequencing analysis system determines to which haplotype in a pair of haplotypes said partial sequencing read corresponds.

35. The method of claim 18, wherein said set of partial sequencing reads comprises DNA sequencing data.

36. The method of claim 18, wherein said set of partial sequencing reads comprises RNA sequencing data.

37. The method of claim 18, wherein said set of partial sequencing reads comprises genomic sequencing data.

24. A method of processing a plurality of nucleic acid molecules derived from a biological sample of a subject, comprising:

(a) providing a sequencing analysis system remotely located with respect to a sequencer, which sequencer sequences said plurality of nucleic acid molecules to generate a set of partial sequencing reads comprising a partial sequencing read having a barcode sequence, wherein said sequencing analysis system comprises a database;

(b) while said sequencer is sequencing said plurality of nucleic acid molecules in (a), receiving by said sequencing analysis system over a network said set of partial sequencing reads from said sequencer, wherein said set of partial sequencing reads is analyzed in real-time by said remote sequencing analysis system while said sequencer is sequencing said plurality of nucleic acid molecules; and

(c) storing said set of partial sequencing reads in said database.

25. The method of claim 24, wherein said set of partial sequencing reads comprises an additional partial sequencing read having an additional barcode sequence.

26. The method of claim 25, wherein said partial sequencing read and said additional partial sequencing read share a common subsequence.

27. The method of claim 25, wherein said partial sequencing read and said additional partial sequencing read are from a common biomolecule.

28. The method of claim 25, wherein (b) comprises aligning said partial sequencing read and said additional partial sequencing read.

29. The method of claim 25, wherein said partial sequencing read and said additional partial sequencing read share a common barcode sequence.

38. The method of claim 24, further comprising processing said partial sequencing read with a reference sequence.

39. The method of claim 24, further comprising processing said partial sequencing read with a reference sequence to identify a mutation in said sample.

40. The method of claim 24, wherein said sequencing analysis system determines a candidate sequence, wherein said candidate sequence incorporates a portion of said partial sequencing read.

41. The method of claim 40, wherein said sequencing analysis system receives a number of partial sequencing reads, and wherein determining said candidate sequence is performed at a rate that increases as said number of partial sequencing reads increases.

42. The method of claim 24, wherein said sequencing analysis system determines to which haplotype in a pair of haplotypes said first partial sequencing read corresponds.

43. The method of claim 24, wherein said set of partial sequencing reads comprises DNA sequencing data.

44. The method of claim 24, wherein said set of partial sequencing reads comprises RNA sequencing data.

45. The method of claim 24, wherein said set of partial sequencing reads comprises genomic sequencing data.