Amplicon preparation and sequencing on solid supports

• US 9,926,598 B2
• Filed: 12/18/2014
• Issued: 03/27/2018
• Est. Priority Date: 01/16/2014
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Xu, Hongxia, Aravanis, Alex, Lin, Shengrong
• Primary Examiner(s): Thomas, David C
• Application Number:
  US14/575,808
• Publication Number:
  US 20150197798A1
• Time in Patent Office: 1,195 Days
• Field of Search: None
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 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 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

The present disclosure relates to the field of molecular biology and more specifically to methods for capturing, amplifying and sequencing target polynucleotides on a solid surface.

Claims

1. A method for amplicon preparation, comprising:

(a) contacting a nucleic acid sample comprising a plurality of target polynucleotides with at least one primer under conditions sufficient for hybridization, said at least one primer containing an adapter;

(b) amplifying by polymerase chain reaction (PCR) said plurality of target polynucleotides to produce a plurality of amplicons;

(c) directly contacting a plurality of target specific capture primers immobilized on a solid support with said plurality of amplicons under conditions sufficient for hybridization to produce a first plurality of immobilized amplicons, said solid support further comprising a plurality of universal capture primers, wherein said adapter is complementary to said plurality of universal capture primers;

(d) extending said plurality of target specific capture primers to produce a plurality of immobilized extension products complementary to said target polynucleotides;

(e) annealing said plurality of universal capture primers to said plurality of said immobilized extension products, and

(f) amplifying by PCR said plurality of immobilized extension products to produce a second plurality of immobilized amplicons, wherein said population of immobilized amplicons comprises a uniformity of 85% or more.

2. The method of claim 1, wherein said plurality of target polynucleotides comprises 10 ng or less input nucleic acid.

3. The method of claim 1, wherein said conditions sufficient for hybridization comprise incubation for 10 minutes or less.

4. The method of claim 1, wherein said directly contacting step (c) comprises a substantially unpurified first plurality of amplicons.

5. The method of claim 1, wherein said amplification of said plurality of target polynucleotides comprises asymmetrical PCR.

6. The method of claim 1, wherein said at least one primer comprises two primers and at least one of said two primers contains said adapter.

7. The method of claim 6, wherein said amplification of said plurality of target polynucleotides comprises multiplex amplification.

8. The method of claim 7, wherein said multiplex amplification comprises a multiplexicity of 180 or more.

9. The method of claim 1, wherein said solid support further comprises a second plurality of universal capture primers.

10. The method of claim 9, wherein said plurality of target specific capture primers further comprise a universal capture primer region.

11. The method of claim 10, wherein said universal capture primer region has a nucleotide sequence corresponding to said second plurality of universal capture primers.

12. The method of claim 1, wherein said plurality of target specific capture primers comprise 200 or more different nucleotide sequences.

13. The method of claim 1, wherein said plurality of target polynucleotides comprise 200 or more different nucleotide sequences.

14. The method of claim 1, wherein said plurality of target polynucleotides comprise a plurality of genomic nucleic acids.

15. The method of claim 1, wherein said amplification of said immobilized extension products comprises bridge amplification.

16. The method of claim 1, wherein said solid support comprises a flow cell.

17. A method for determining the presence of a cancer associated gene, comprising:

(a) contacting a nucleic acid sample comprising a plurality of target polynucleotides with at least one primer under conditions sufficient for hybridization, said at least one primer containing an adapter;

(b) amplifying by polymerase chain reaction (PCR) said plurality of target polynucleotides to produce a plurality of amplicons;

(c) directly contacting a plurality of target specific capture primers specific to one or more different cancers immobilized on a solid support with said plurality of amplicons under conditions sufficient for hybridization to produce a first plurality of immobilized amplicons, said solid support further comprising a plurality of universal capture primers, wherein said adapter is complementary to said plurality of universal capture primers;

(d) extending said plurality of target specific capture primers to produce a plurality of immobilized extension products complementary to said target polynucleotides;

(e) annealing said plurality of universal capture primers to said plurality of said immobilized extension products;

(f) amplifying by PCR said plurality of immobilized extension products to produce a second plurality of immobilized amplicons, wherein said population of immobilized amplicons comprises a uniformity of 85% or more, and

(g) sequencing said second plurality of immobilized extension products to determine the presence or absence of a cancer associated gene.

18. A method for determining a true nucleic acid sequence variant, comprising:

(a) contacting a nucleic acid sample comprising a plurality of target polynucleotides with gene specific forward and reverse primers under conditions sufficient for hybridization, each species of said gene specific forward primer comprising a unique sequence index and an adapter;

(b) amplifying by polymerase chain reaction (PCR) said plurality of target polynucleotides to produce a plurality of amplicons;

(c) directly contacting a plurality of target specific capture primers immobilized on a solid support with said plurality of amplicons under conditions sufficient for hybridization to produce a first plurality of immobilized of amplicons, said solid support further comprising a plurality of universal capture primers, wherein said adapter is complementary to said plurality of universal capture primers;

(d) extending said plurality of target specific capture primers to produce a plurality of immobilized extension products complementary to said target polynucleotides;

(e) annealing said plurality of universal capture primers to said plurality of said immobilized extension products;

(f) amplifying by PCR said plurality of immobilized extension products to produce a second plurality of immobilized amplicons, wherein said second plurality of immobilized amplicons comprises a uniformity of 85% or more;

(g) sequencing said second plurality of immobilized amplicons, and

(h) eliminating random sequence errors for one or more target polynucleotide by comparing three or more nucleotide sequences at a variant position for a target polynucleotide species, wherein said target polynucleotide species are identified by said unique sequence index to thereby determine a true nucleotide sequence variant in said one or more target polynucleotides.