Methods and systems for sequencing long nucleic acids

Patent metadata

Field	Value
Publication number	US-10801062-B2
Application number	US-201214009089-A
Country	US
Kind code	B2
Filing date	Apr 2, 2012
Priority date	Apr 1, 2011
Publication date	Oct 13, 2020
Grant date	Oct 13, 2020

Abstract

The present invention provides methods and systems for sequencing long nucleic acid fragments. In one aspect of the invention, methods, systems and reagent kits are provided for sequencing nucleic acid target sequences. Some embodiments of the methods, systems and reagent kits are particularly suitable for sequencing a large number of fragments, particularly long fragments.

First claim

What is claimed is:

1. A method for sequencing a target nucleic acid, comprising the steps of:
(a) providing a first extension primer hybridized with said target nucleic acid;
(b) extending said first extension primer, said extending in step (b) comprises a first controlled extension comprising (i) contacting said first extension primer with a first set of nucleotides comprising three different unmodified nucleotides to a first defined length, or (ii) contacting said first extension primer with said first set of nucleotides comprising said three different unmodified nucleotides and a reversible terminator nucleotide to said first defined length, thereby said extending in step (b) produces an extended product comprising said first extension primer and an extended portion, wherein sequence of said extended portion is unknown;
(c) using the extended product obtained in step (b) as a first sequencing primer from which to generate a first sequence read, and sequencing a first region of said target nucleic acid;
(d) removing at least a part of said first sequence read;
(e) providing a second extension primer hybridized with said target nucleic acid;
(f) extending said second extension primer to a second defined length; and
(g) sequencing a second region of said target nucleic acid, wherein said sequencing in (g) comprises providing the extended product obtained in step (f) as a second sequencing primer from which to generate a second sequence read, and wherein said second region is different from said first region.

2. The method of claim 1 , wherein said removing in step (d) comprises enzymatic digestion of said first sequence read.

3. The method of claim 1 , wherein said removing in step (d) comprises exonuclease digestion and wherein a base that is resistant to exonuclease digestion is incorporated to a position in said first sequence read during said sequencing in step (c).

4. The method of claim 1 , wherein said first and second extension primers are the same.

5. The method of claim 1 , wherein said first and second extension primers are different.

6. The method of claim 1 , wherein said extending in step (b) comprises said first controlled extension comprising contacting said first extension primer with said first set of nucleotides comprising said three different unmodified nucleotides, wherein said extending in step (b) further comprises a second controlled extension after said first controlled extension, wherein said second controlled extension uses a second set of nucleotides comprising another three different unmodified nucleotides, wherein said extended product in steps (b) further comprises another extended portion after said extended portion, wherein before said second controlled extension, said first set of nucleotides used in said first controlled extension are removed.

7. The method of claim 6 , wherein said first set of nucleotides used in said first controlled extension is different than said second set of nucleotides used in said second controlled extension.

8. The method of claim 6 , wherein said first set of nucleotides are removed by washing and a nucleotide degrading enzyme prior to said second controlled extension.

9. The method of claim 1 , wherein said first controlled extension is performed with said first set of nucleotides and said reversible terminator nucleotide to incorporate said reversible terminator nucleotide, wherein before a subsequent controlled extension, said reversible terminator nucleotide incorporated is deblocked.

10. The method of claim 1 , wherein the sequence of said target nucleic acid is determined by assembling said first, second, and optionally additional sequence reads.

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

12. The method of claim 11 , wherein said substrate is a flow cell.

13. The method of claim 11 , wherein said substrate comprises glass.

14. The method of claim 11 , wherein said target nucleic acid is attached to said substrate via a capture probe.

15. The method of claim 1 , wherein said first and second sequence reads start at positions that are at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 175, or 200 bases apart on said target nucleic acid.

Assignees

• Zhou Wei
• Mei Rui
• Crnogorac Filip
• Liao Guochun
• Lucas Julian
• Centrillion Tech Holdings Corp

Inventors

• Zhou Wei
• Mei Rui
• Crnogorac Filip
• Liao Guochun
• Lucas Julian

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q2527/113

  Time · CPC title

• C12Q2525/185

  incorporating bases where the precise position of the bases in the nucleic acid string is important · CPC title