Methods and compositions for preparing sequencing libraries

• US 10,443,087 B2
• Filed: 06/12/2015
• Issued: 10/15/2019
• Est. Priority Date: 06/13/2014
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Rigatti, Roberto, Gormley, Niall Anthony
• Primary Examiner(s): Boesen, Christian C
• Application Number:
  US15/318,258
• Publication Number:
  US 20170130260A1
• Time in Patent Office: 1,586 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Embodiments provided herein relate to methods and compositions for next generation sequencing. Some embodiments include the preparation of a template library from a target nucleic acid in contact with a surface, and sequencing the library on the surface.

Claims

1. A method of preparing a population of target nucleic acids for sequencing comprising:

(a) providing a substrate having a surface comprising capture probes;

(b) contacting the surface with a reaction volume comprising a plurality of template nucleic acids and transposomes, wherein each transposome comprises a transposon sequence and a transposase, wherein the template nucleic acids are prepared by contacting a target nucleic acid with a plurality of transposomes; and

(c) associating the template nucleic acids with the capture probes in the presence of a recombinase.

2. The method of claim 1, comprising (d) sequencing the associated template nucleic acids.

3. The method of claim 2, comprising amplifying the associated template nucleic acids after (c) and before (d).

4. The method of claim 3, wherein the amplifying comprises bridge amplification.

5. The method of claim 1, wherein step (a) comprises providing a sample comprising the target nucleic acid and the transposomes.

6. The method of claim 5, wherein the sample comprises a polymerase and/or a ligase.

7. The method of claim 1, wherein the template nucleic acids are prepared by contacting the target nucleic acid with the plurality of transposomes in the presence of the surface.

8. The method of claim 1, wherein the probes comprise nucleic acids.

9. The method of claim 8, wherein the capture probes comprise the recombinase.

10. The method of claim 1, wherein the template nucleic acids comprise an affinity moiety selected from the group consisting of biotin, avidin, and streptavidin.

11. The method of claim 1, wherein the transposon sequence comprises a fragmentation site and wherein the method comprises the step of cleaving the fragmentation site.

12. The method of claim 11, wherein the fragmentation site is cleaved after step (c).

13. The method of claim 1, wherein the transposase is removed by contacting the transposase with a protease or SDS.

14. The method of claim 1, wherein at least one transposome is different from at least one other transposome.

15. The method of claim 1, comprising amplifying the associated template nucleic acids after (c) using an amplification technique facilitated by the recombinase.

16. The method of claim 15, wherein the recombinase binds regions of double-stranded nucleic acids comprising non-complementary strands during the amplifying.

17. The method of claim 1, wherein the capture probes are immobilized on the substrate.