Methods for selecting and amplifying polynucleotides

• US 10,597,653 B2
• Filed: 04/17/2017
• Issued: 03/24/2020
• Est. Priority Date: 03/10/2008
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Sabot, Andrea, Rigatti, Roberto, Shen, Min-Jui Richard
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US15/489,549
• Publication Number:
  US 20170283794A1
• Time in Patent Office: 1,072 Days
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• 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 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• 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 invention provides methods for generating a nucleic acid library. Target-specific primer-probes are hybridized to a target nucleic acid fragment to create a hybridization product. Each of the target-specific primer-probes comprises a target-specific sequence and a first adaptor sequence. The target nucleic acid fragment comprises a target genomic region of interest, wherein said target genomic region of interest comprises an exon of a gene and the target-specific primer-probes are tiled across the exon of the gene. The target nucleic acid fragment further comprises a second adaptor sequence different from said first adaptor sequence. Following hybridization, the target-specific primer-probes are extended to create double-stranded extension products and further amplified, thereby generating the nucleic acid library.

Claims

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

a) hybridizing target-specific primer-probes to a target nucleic acid fragment to create a hybridization product;

b) extending said target-specific primer-probes to create double-stranded extension products; and

c) amplifying said extension products, thereby generating the nucleic acid library;

wherein each of said target-specific primer-probes comprises a target-specific sequence and a first adaptor sequence;

said target nucleic acid fragment comprises a target genomic region of interest comprising an exon of a gene; and

said target nucleic acid fragment comprises a second adaptor sequence different from said first adaptor sequence.

2. The method of claim 1, wherein said target nucleic acid fragment comprises DNA.

3. The method of claim 1, wherein said target nucleic acid fragment comprises RNA.

4. The method of claim 1, wherein said hybridization, extension and said amplification steps are performed directly inside a next-generation DNA sequencer.

5. The method of claim 1, wherein said target-specific primer-probes are selectively hybridizable to said target genomic region of interest.

6. The method of claim 1, wherein said amplification comprises bridge amplification.

7. The method of claim 1, further comprising sequencing said amplified extension products.

8. The method of claim 7, wherein said sequencing comprises use of a parallel sequencing platform.

9. The method of claim 8, wherein said first adaptor sequence comprises a sequencing platform-specific sequence for binding to a solid support of said sequencing platform.

10. The method of claim 1, wherein said first adaptor sequence comprises a binding site for a sequencing primer.

11. The method of claim 1, further comprising ligating said second adaptor sequence to said target nucleic acid fragment.

12. The method of claim 11, wherein said second adaptor sequence is ligated to one end but not both ends of said target nucleic acid fragment.

13. The method of claim 1, wherein said second adaptor sequence comprises an index sequence.

14. The method of claim 13, wherein said index sequence allows a source of said target nucleic acid fragment to be identified.

15. The method of claim 1, further comprising hybridizing a target-specific primer-probe to a sequence that flanks said exon.

16. The method of claim 1, further comprising hybridizing said target-specific primer-probes to each strand of said exon.

17. The method of claim 1, further comprising performing targeted resequencing of said gene in a plurality of samples.

18. The method of claim 1, further comprising hybridizing a plurality of target-specific primer-probes to a plurality of target nucleic acid fragments.

19. The method of claim 1, wherein said gene is a ribosomal gene.

20. The method of claim 19, wherein said ribosomal gene is 16S or 18S rRNA gene.