Methods and systems for sequencing long nucleic acids

• US 9,689,032 B2
• Filed: 08/19/2013
• Issued: 06/27/2017
• Est. Priority Date: 04/01/2011
• Status: Active Grant

• Current Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Original Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Inventors: Lucas, Julian, Mei, Rui, Liao, Guochun, Zhou, Wei, Crnogorac, Filip
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/970,321
• Publication Number:
  US 20140065604A1
• Time in Patent Office: 1,408 Days
• Field of Search: 435 611, 506 7
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

Abstract

The present invention provides methods and systems for sequencing long nucleic acid fragments.

Claims

1. A method for sequencing a target nucleic acid, comprising:

(a) sequencing one or more bases of a target nucleic acid by extending a first sequencing primer hybridized to said target nucleic acid to generate a first primer extension product, thereby obtaining a first sequence read;

(b) releasing said first primer extension product from said target nucleic acid;

(c) hybridizing a second sequencing primer to said target nucleic acid;

(d) generating a second primer extension product by extending said second sequencing primer through limited extension without the use of labeled nucleotides;

(e) sequencing one or more bases of said target nucleic acid by further extending said second primer extension product to generate a third primer extension product, thereby obtaining a second sequence read; and

(f) determining a sequence of said target nucleic acid by assembling said first sequence read and said second sequence read.

2. The method of claim 1, wherein said first sequencing primer and said second sequencing primer are the same.

3. The method of claim 1, wherein said first sequencing primer and said second sequencing primer are different.

4. The method of claim 1, wherein said limited extension is carried out by pulse extension.

5. The method of claim 4, wherein said pulse extension is carried out by allowing the extending reaction to last 30 to 60 seconds.

6. The method of claim 1, wherein said limited extension is carried out by using a nucleic acid polymerase and one or more sets of nucleotides, wherein each set comprises not more than three different nucleotides.

7. The method of claim 6, wherein said extending is with more than one set of nucleotides.

8. The method of claim 7, wherein said set of nucleotides comprises three different nucleotides.

9. The method of claim 6, wherein prior to a subsequent addition of a set of nucleotides a washing step is performed.

10. The method of claim 6, wherein prior to a subsequent addition of a set of nucleotides a nucleotide degradation step is performed.

11. The method of claim 1, further comprising repeating steps (b) to (e) by releasing the primer extension product generated in step (e), thereby obtaining one or more additional sequence reads.

12. The method of claim 11, wherein the sequence of said target nucleic acid is determined by assembling said first, second, and at least one of the one or more additional sequence reads.

13. The method of claim 1, wherein said sequencing in (a) is by extending the first sequencing primer using a labeled reversible terminator or said sequencing in (e) is by further extending the second primer extension product using a labeled reversible terminator.

14. The method of claim 1, wherein said target nucleic acid is attached to a substrate.

15. The method of claim 14, wherein said substrate is a flat surface or bead.

16. The method of claim 14, wherein said substrate is a flow cell.

17. The method of claim 14, wherein said substrate comprises glass.

18. The method of claim 14, wherein said target nucleic acid is attached to said substrate via a capture probe.

19. The method of claim 1, further comprising analyzing said sequencing results to provide a diagnosis, prognosis, or theranosis for a subject.

20. The method of claim 1, comprising sequencing a plurality of target nucleic acids.

23. The method of claim 1 or claim 21, wherein said sequencing in (a) and said sequencing in (e) comprise sequencing by synthesis.

25. The method of claim 1 or claim 21, wherein said first sequence read and said second sequence read are each at least 5 bases long.

26. The method of claim 1 or claim 21, wherein said first sequence read and said second sequence read are each at least 10 bases long.

21. A method for sequencing a target nucleic acid, comprising:

(a) sequencing one or more bases of a target nucleic acid by extending a first sequencing primer hybridized to said target nucleic acid to generate a first primer extension product, thereby obtaining a first sequence read;

(b) releasing said first primer extension product from said target nucleic acid;

(c) hybridizing a second sequencing primer to said target nucleic acid;

(d) generating a second primer extension product by extending said second sequencing primer by a first number of nucleotides through limited extension without the use of labeled nucleotides;

(e) sequencing one or more bases of said target nucleic acid by further extending said second primer extension product to generate a third primer extension product, thereby obtaining a second sequence read;

(f) releasing said third primer extension product from said target nucleic acid;

(g) hybridizing a third sequencing primer to said target nucleic acid;

(h) generating a fourth primer extension product by extending said third sequencing primer by a second number of nucleotides through limited extension without the use of labeled nucleotides;

(i) sequencing one or more bases of said target nucleic acid by further extending said fourth primer extension product to generate a fifth primer extension product, thereby obtaining a third sequence read; and

(j) determining a sequence of said target nucleic acid by assembling said first sequence read, said second sequence read, and said third sequence read,

wherein said first number of nucleotides is different from said second number of nucleotides.

22. The method of claim 21, wherein said first number of nucleotides and said second number of nucleotides are different by at least 10 nucleotides.

24. The method of claim 21, wherein said target nucleic acid is immobilized on a solid support.