Methods and systems for sequencing long nucleic acids

• US 10,174,368 B2
• Filed: 06/23/2017
• Issued: 01/08/2019
• Est. Priority Date: 09/10/2009
• Status: Active Grant

• Current Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Original Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Inventors: Zhou, Wei, Mei, Rui, Matsuzaki, Hajime
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/631,414
• Publication Number:
  US 20180010180A1
• Time in Patent Office: 564 Days
• 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 2521/501 Ligase
• 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 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

Abstract

The present invention provides methods and systems for sequencing long nucleic acid fragment. The present invention also provides a method of sequencing a target polynucleotide with fewer probes. Further, the present invention provides a method of sequencing a target polynucleotide with longer reads. Locus-specific, ligation-assisted sequencing/genotyping method and ligation-captured sequencing method are also provided in the present invention. The methods of the present invention allow low-cost, high-throughput and accurate sequencing of nucleic acids.

Claims

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

removing the target nucleic acid from a partial copy of the target nucleic acid hybridized to the target nucleic acid, wherein the partial copy of the target nucleic acid comprises;

(i) a capture probe from a plurality of capture probes;

(ii) a first solution probe from a plurality of first solution probes;

(iii) a second solution probe from a plurality of second solution probes; and

(iv) a third solution probe from a plurality of third solution probes; and

sequencing one or more bases of the partial copy of the target nucleic acid by extending a primer hybridized to a 3′

end of the partial copy of the target nucleic acid to generate a first extension product, thereby obtaining a first sequence read to determine the sequence of the partial copy of the target nucleic acid by performing a sequencing reaction, wherein the target nucleic acid is from a plurality of target nucleic acid fragments; and

wherein the capture probe is ligated to the first solution probe, the first solution probe is ligated to both the capture probe and the second solution probe, the second solution probe is ligated to both the first and third solution probes, and the third solution probe comprises a universal primer sequence at the 3′

end complementary to the primer.

2. The method of claim 1, prior to said removing, further comprising:

(x1) selectively removing a first cap from a first probe by cleaving a cleavage site at a 3′

end of the first probe, wherein the first probe comprises;

(i) a capture probe from a plurality of capture probes; and

(ii) a first solution probe from a plurality of first solution probes, the first solution probe hybridized to the target nucleic acid via sequence complementary to a second region of a known sequence of the target nucleic acid, each of the plurality of first solution probes comprising the first cap on the 3′

end linked via the cleavage site;

(x2) hybridizing the second solution probe to the target nucleic acid, wherein the second solution comprises sequence complementary to a third region of the known sequence of the target nucleic acid, wherein the second solution probe is in solution;

(x3) ligating the second solution probe to the first solution probe hybridized to the target nucleic acid;

(x4) hybridizing the third solution probe to the target nucleic acid, wherein the third solution comprises sequence complementary to a fourth region of the known sequence of the target nucleic acid, wherein the third solution probe is in solution;

(x5) ligating the third solution probe to the second solution probe, thereby forming the partial copy of the target nucleic acid hybridized to the target nucleic acid.

3. The method of claim 2, prior to said removing, further comprising:

adding a second cap to any of the plurality of capture probes not ligated to any of the plurality of first solution probes.

4. The method of claim 3, prior to said adding the second cap, further comprising:

ligating the capture probe and the first solution probe, thereby forming the first probe.

9. The method of claim 2, wherein the first cap comprises a capping dye.

10. The method of claim 2, wherein the first cap comprises a nucleotide reversible terminator.

11. The method of claim 2, wherein each capture probe of the plurality of capture probes comprises 50-150 nucleotides.

12. The method of claim 2, wherein each first solution probe of the plurality of first solution probes comprises 5-50 nucleotides.

13. The method of claim 2, wherein each second solution probe of the plurality of second solution probes and/or each third solution probe of the plurality of third solution probes comprise 5-50 nucleotides.

14. The method of claim 1, wherein said sequencing reaction comprises a base extension reaction in the presence of labeled nucleotides.

5. The method of 4, prior to said ligating the capture probe and the first solution probe, further comprising:

hybridizing the target nucleic acid to the capture probe and the first solution probe, wherein the capture probe comprises sequence complementary to a first region of a known sequence of the target nucleic acid, wherein the first solution probe is in solution,- and wherein said hybridizing is configured to permit ligation of the capture probe and the first solution probe.

6. The method of claim 5, wherein each capture probe of the plurality of capture probes is attached to a solid support, each first solution probe of the plurality of first solution probes is in solution.

7. The method of claim 6, wherein each first solution probe of the plurality of first solution probes each second solution probe of the plurality of second solution probes and/or each third solution probe of the plurality of third solution probes comprise at least one base that is degenerate.

8. The method of claim 5, further comprising purposefully removing any of the plurality of first solution probes and any of the plurality of target nucleic acid fragments not engaged in specific hybridization, wherein said purposefully removing comprises performing one or more washes under stringent conditions.