Methods and compositions using one-sided transposition

• US 10,577,603 B2
• Filed: 06/26/2015
• Issued: 03/03/2020
• Est. Priority Date: 06/30/2014
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Steemers, Frank J., Fisher, Jeffrey S., Gunderson, Kevin L., Amini, Sasan, Gloeckner, Christian
• Primary Examiner(s): Havlin, Robert H
• Application Number:
  US15/322,432
• Publication Number:
  US 20180201925A1
• Time in Patent Office: 1,712 Days
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• 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 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

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 using one-sided transposition, and sequencing the template library.

Claims

1. A method of preparing a sequencing library with captured contiguity information from a double-stranded target nucleic acid comprising:

(a) providing a plurality of transposomes, each transposome comprising a transposase and a transposon nucleic acid, wherein the transposase is configured to nick only one strand of the double stranded target nucleic acid;

(b) contacting the target nucleic acid with the plurality of transposomes such that the target nucleic acid is nicked at a plurality of sites of the target nucleic acid and single transposon nucleic acids are attached to at least one the nicked target nucleic acid to generate transposed nucleic acids; and

(c) extending the transposed nucleic acids to generate a plurality of extended nucleic acids which comprises overlapping sequences in a linear representation of a single target nucleic acid sequence, thereby obtaining a library of modified nucleic acids with captured contiguity information.

2. The method of claim 1, further comprising capturing the modified nucleic acids on a surface.

3. The method of claim 1, wherein the transposomes that are contacted with the target nucleic acids in (b) are attached to a surface, thereby capturing the target nucleic acids on the surface.

4. The method of claim 2, further comprising sequencing the captured nucleic acids on the surface.

5. The method of claim 4, wherein a proximity of sequence information obtained from two captured nucleic acids in the linear representation of the target nucleic acid sequence is indicative of the proximity of the captured nucleic acids on the surface.

6. The method of claim 5, wherein captured nucleic acids in closer proximity to one another on the surface comprise sequences in closer proximity in the representation of the target nucleic acid sequence compared to sequences in the representation of the target nucleic acid sequence of captured nucleic acids in less close proximity to one another on the surface.

7. The method of claim 1, wherein the linear representation of the target nucleic acid sequence comprises a haplotype representation.

8. The method of claim 1, wherein the transposase comprises a one-sided transposase activity.

9. The method of claim 1, wherein one or more transposon nucleic acids are non-functional, wherein the 3′

end of the non-functional transposon nucleic acid is selected from the group consisting of a dideoxy group, a spacer group, an amine group, an alkyl group, an aryl group, a phosphate group, a thiol group, a reverse nucleotide, an azido group, a sulfate group, and a biotin group.

10. The method of claim 9, wherein a ratio of transposon nucleic acids comprising non-functional transposon nucleic acids to functional transposon nucleic acids is greater than or equal to 1:

1.

11. The method of claim 2, further comprising amplifying the captured nucleic acids on the surface.

12. The method of claim 2, wherein the surface comprises a plurality of capture probes.

13. The method of claim 1, wherein the transposon nucleic acid comprises a sequence selected from the group consisting of an anchor site, a barcode, a sequencing primer site, an amplification primer site, and a reporter tag.

14. The method of claim 1, wherein at least one transposome comprises two transposon nucleic acids.

15. The method of claim 1, wherein the plurality of transposomes comprise at least two different transposon nucleic acids.

16. The method of claim 1, wherein after contacting the target nucleic acid with the transposomes, the transposases are removed from the target nucleic acid by treating the transposases with SDS, urea, heat, or a protease.

17. The method of claim 1, wherein the target nucleic acid is selected from the group consisting of genomic DNA, fragments of genomic DNA, and cDNA.

18. The method of claim 2, wherein the surface is on a substrate selected from the group consisting of a bead, slide, flow cell, channel, dip-stick, and well.

19. The method of claim 2, wherein the surface comprises at least about 10,000 captured nucleic acids per mm².

20. The method of claim 1, further comprising combinatorial barcoding, wherein the transposon comprises a first set of barcodes;

the first set of barcodes are introduced to the target nucleic acid during transposition to generate transposed target nucleic acids comprising first set of barcodes;

the transposed target nucleic acids are pooled to generate a first pool of transposed target nucleic acids;

a second set of barcodes are introduced to the first pool of transposed target nucleic acid to generate target nucleic acids comprising first and second sets of barcodes;

target nucleic acids comprising first and second sets of barcodes;

are pooled to generate a second pool of transposed target nucleic acid;

optionally repeating the steps of introducing additional barcodes and pooling to generate a library of barcoded target nucleic acids.

21. The method of claim 1, wherein step (c) is performed in solution.