Methods of sequencing with linked fragments

• US 10,000,799 B2
• Filed: 11/02/2015
• Issued: 06/19/2018
• Est. Priority Date: 11/04/2014
• Status: Active Grant

• Current Assignee: NCAN Genomics Incorporated
• Original Assignee: Boreal Genomics Inc.
• Inventors: Despotovic, Milenko, Pel, Joel, Marziali, Andrea
• Primary Examiner(s): Thomas, David C
• Application Number:
  US14/930,227
• Publication Number:
  US 20160122814A1
• Time in Patent Office: 960 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

The invention generally relates to sequencing library preparation methods. In certain embodiments, two template nucleic acids are joined together by a linking molecule, such as a PEG derivative. The linked template nucleic acids is amplified, creating linked amplicons.

Claims

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

annealing to a solid support a complex comprising two nucleic acid templates joined by a linker, wherein the linker comprises a molecule that is not selected from the group consisting of guanine, cytosine, adenine, uracil or thymine;

amplifying the complex to generate amplification products; and

sequencing the amplification products.

2. The method of claim 1, wherein the linker is selected from the group consisting of a polyethylene glycol derivative, an oligosaccharide, a lipid, a hydrocarbon, a polymer, an antibody, and a protein.

3. The method of claim 1, wherein the linker comprises an inverted or modified base.

4. The method of claim 1, wherein the linker is removed prior to the amplifying step.

5. The method of claim 1, wherein the method further comprises introducing an adapter to the complex.

6. The method of claim 1, wherein the method further comprises the step of introducing primers to the complex.

7. The method of claim 1, wherein the amplifying step comprises bridge amplification.

8. The method of claim 1, further comprising attaching the amplification products to the solid support.

9. The method of claim 8, wherein the amplification products form a cluster on the solid support.

10. The method of claim 1, wherein the sequencing step comprises sequencing-by-synthesis.

11. The method of claim 10, wherein said sequencing-by-synthesis comprises addition of fluorescently-labeled nucleotides.

12. A method of sequencing a nucleic acid, the method comprising:

attaching at least two copies of a nucleic acid fragment, forming a unit;

joining at least two units with a linker to form a complex, wherein the linker comprises a molecule that is not selected from the group consisting of guanine, cytosine, adenine, uracil or thymine;

annealing the complex to a solid support;

amplifying the complex; and

sequencing the amplification products.

13. The method of claim 12, wherein the linker is selected from the group consisting of a polyethylene glycol derivative, an oligosaccharide, a lipid, a hydrocarbon, a polymer, an antibody, and a protein.

14. The method of claim 12, wherein the linker comprises an inverted or modified base.

15. The method of claim 12, wherein the method further comprises introducing an adapter to the complex.