Methods and compositions for targeted nucleic acid sequencing

• US 10,100,355 B2
• Filed: 12/05/2014
• Issued: 10/16/2018
• Est. Priority Date: 12/09/2013
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Pantoja, Rigo, Hu, Marie, Bomati, Erin, He, Molly
• Primary Examiner(s): Woolwine, Samuel C
• Assistant Examiner(s): Oyeyemi, Olayinka A
• Application Number:
  US14/562,307
• Publication Number:
  US 20150159212A1
• Time in Patent Office: 1,411 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Embodiments provided herein relate to methods and compositions for obtaining nucleic acid sequence information. Some embodiments provided herein include methods and compositions for preparing nucleic acid libraries. In some embodiments, such nucleic acid libraries are useful for targeted nucleic acid sequencing.

Claims

1. A method for preparing a substrate-bound anchor probe comprising a sequence of a target nucleic acid comprising:

(a) providing a nucleic acid library comprising a plurality of different nucleic acids, wherein the nucleic acid library comprises a double-stranded target nucleic acid;

(b) hybridizing a first adapter probe with the target nucleic acid in the presence of a recombinase,

wherein the first adapter probe comprises a first portion complementary to a substrate probe and a second portion complementary to at least a portion of the double-stranded target nucleic acid, and

wherein the recombinase promotes hybridizing the second portion with the double-stranded target nucleic acid;

(c) extending the hybridized first adapter probe;

(d) hybridizing the extended adapter probe with the substrate probe, wherein the substrate probe is immobilized on a first substrate; and

(e) extending the hybridized substrate probe to form a substrate-bound anchor probe comprising the sequence of the target nucleic acid.

2. The method of claim 1, further comprising amplifying the substrate-bound anchor probe, the amplifying comprising:

(f) hybridizing a second adapter probe with the substrate-bound anchor probe, wherein the second adapter probe comprises a first portion complementary to a capture probe and a second portion complementary to at least a portion of the substrate-bound anchor probe;

(g) extending the hybridized, substrate-bound anchor probe to form an extended anchor probe;

(h) hybridizing the extended anchor probe with the capture probe, wherein the capture probe is immobilized on a second substrate; and

(i) extending the capture probe to form an immobilized, modified capture probe.

3. The method of claim 1, wherein (c) comprises polymerase extension.

4. The method of claim 3, wherein the polymerase extension comprises a polymerase chain reaction.

5. The method of claim 1, wherein (e) comprises polymerase extension.

6. The method of claim 1, wherein (e) comprises ligation of a nucleic acid complementary to the extended adapter probe with the substrate probe.

7. The method of claim 1, wherein the first adapter probe comprises a sequence selected from the group consisting of a locus-specific sequence and an allele-specific sequence.

8. The method of claim 2, wherein the second adapter probe comprises a sequence selected from the group consisting of a locus-specific sequence and an allele-specific sequence.

9. The method of claim 1, wherein the double-stranded target nucleic acid comprises genomic DNA.

10. The method of claim 1, wherein the recombinase is RecA.

11. The method of claim 1, wherein the first substrate comprises a bead.

12. The method of claim 11, wherein the bead is associated with an individual site of a substrate.

13. The method of claim 12, wherein the site is configured to have a single associated bead.

14. The method of claim 2, wherein the first and the second substrate are the same substrate.

15. The method of claim 1, wherein the nucleic acids in the starting nucleic acid library comprise one or more sequences selected from mosaic elements, indices, and sequencing adapters.

16. The method of claim 1, wherein the nucleic acid library is produced by tagging and fragmenting activities of a transposase.

17. A method of detecting a plurality of nucleic acid sequences comprising:

obtaining a plurality of immobilized, modified capture probes prepared according to claim 2; and

detecting the immobilized, modified capture probes, thereby detecting the plurality of target nucleic acid sequences.

18. A method of preparing a nucleic acid library comprising:

(a) providing a plurality of anchor probes, wherein the anchor probes are each immobilized on a substrate;

(b) hybridizing a plurality of double-stranded target nucleic acids with the plurality of anchor probes in the presence of a recombinase under conditions wherein the recombinase promotes the hybridizing with the target nucleic acids;

(c) hybridizing the plurality of double-stranded target nucleic acids with a plurality of ligation probes in the presence of a recombinase under conditions wherein the recombinase promotes the hybridizing with the target nucleic acids;

(d) extending the anchor probes or the ligation probes; and

(e) ligating the anchor probes to the ligation probes to form a plurality of different extended ligated probes, wherein the extended ligated probes are immobilized to the substrate, thereby preparing the library of nucleic acids.

20. The method of claim 18, wherein the substrate is a flow cell.

21. The method of claim 18, wherein the anchor probe and the extended ligated probe are covalently immobilized to the substrate.

22. The method of claim 18, further comprising amplifying the extended ligated probes by cluster amplification.

19. A method of detecting a nucleic acid comprising:

(a) providing an anchor probe, wherein the anchor probe is immobilized on a substrate;

(b) hybridizing a double-stranded target nucleic acid with the anchor probe in the presence of a recombinase under conditions wherein the recombinase promotes the hybridizing with the target nucleic acid;

(c) hybridizing the double-stranded target nucleic acid with a ligation probe in the presence of a recombinase under conditions wherein the recombinase promotes the hybridizing with the target nucleic acid;

(d) extending the anchor probe or the ligation probe;

(e) ligating the anchor probe to the ligation probe to form an extended ligated probe, wherein the extended ligation probe is immobilized to the substrate; and

(f) detecting the extended ligated probe, thereby detecting the nucleic acid.

23. The method of claim 19, wherein the detecting is by next-generation sequencing.