Direct capture, amplification and sequencing of target DNA using immobilized primers

Patent metadata

Field	Value
Publication number	US-10072283-B2
Application number	US-201414300048-A
Country	US
Kind code	B2
Filing date	Jun 9, 2014
Priority date	Sep 24, 2010
Publication date	Sep 11, 2018
Grant date	Sep 11, 2018

Abstract

Certain embodiments provide a method for capturing a genomic fragment. The method may comprise: obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides; hybridizing a first member of the first population of surface-bound oligonucleotides to a selection oligonucleotide comprising a region that hybridizes with the first member and a region that contains a genomic sequence; extending the first member of the first population of surface-bound oligonucleotides to produce a support-bound selection primer that comprises a sequence that is complementary to the genomic sequence; hybridizing the support-bound selection primer to a nucleic acid fragment comprising the genomic sequence; extending the support-bound selection primer to produce an extension product that contains a sequence that flanks the genomic sequence, e.g., in a genome; and amplifying the extension product on the substrate.

First claim

What is claimed is:

1. A method for generating a nucleic acid library, said method comprising:
a) hybridizing target-specific primer-probes comprising a target-specific sequence and a first adaptor sequence to a target nucleic acid fragment comprising i) a target genomic region of interest comprising an exon of a cancer gene and ii) a second adaptor sequence different from said first adaptor sequence to create hybridization products in which said target-specific primer-probes are tiled across said exon of said cancer gene;
b) extending said target-specific primer-probes to create double-stranded extension products; and
c) amplifying said extension products.

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

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

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

5. The method of claim 4 , wherein said sequencing comprises use of a parallel sequencing platform.

6. The method of claim 1 , wherein said amplification comprises bridge polymerase chain reaction (PCR).

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

8. The method of claim 1 , wherein said first adaptor sequence comprises a sequencing platform-specific sequence for binding to a solid support of a sequencing platform.

9. The method of claim 1 , wherein said second adaptor sequence is ligated to one end but not both ends of said target nucleic acid fragment.

10. The method of claim 1 , wherein said second adaptor sequence comprises a barcode sequence.

11. The method of claim 10 , wherein said barcode sequence allows a source of said target nucleic acid fragment to be identified.

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

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

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

15. The method of claim 1 , further comprising extracting said target nucleic acid fragment from tumor tissue.

16. A method for targeted sequencing, comprising:
a) hybridizing target-specific primer-probes to a single-stranded DNA fragment from a tissue sample, wherein said target-specific primer-probes comprise an adaptor sequence and a sequence specific for a cancer gene, and said target-specific primer-probes are tiled across an exon of said cancer gene;
b) extending said target-specific primer-probes to create double-stranded extension products; and
c) sequencing said extension products.

17. The method of claim 16 , wherein said single-stranded DNA fragment comprises a target genomic region and a second adaptor sequence, and wherein said second adaptor sequence is different than said adaptor sequence of said target-specific primer-probes.

18. The method of claim 17 , wherein said target genomic region comprises said cancer gene.

19. The method of claim 18 , wherein said target genomic region comprises said exon of said cancer gene.

20. The method of claim 16 , further comprising hybridizing a target-specific primer-probe to a sequence that flanks said exon of said cancer gene.

21. The method of claim 16 , wherein said adaptor sequence comprises a sequencing platform-specific sequence.

22. The method of claim 21 , wherein said sequencing platform comprises a next-generation DNA sequencer.

23. The method of claim 17 , wherein said second adaptor sequence is ligated to one end but not both ends of said single-stranded DNA fragment.

24. The method of claim 17 , wherein said second adaptor sequence comprises a barcode sequence.

25. The method of claim 24 , wherein said barcode sequence allows a source of said single-stranded DNA fragment to be identified.

26. The method of claim 16 , wherein said sequencing comprises use of a parallel sequencing platform.

27. The method of claim 16 , further comprising hybridizing a plurality of target-specific primer-probes to a plurality of single-stranded DNA fragments.

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

29. The method of claim 1 , wherein said cancer gene comprises a somatic mutation.

30. The method of claim 1 , wherein said cancer gene comprises KRAS.

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

32. The method of claim 1 , wherein said target nucleic acid fragment is from a formalin-fixed paraffin-embedded sample.

33. The method of claim 1 , further comprising sequencing said amplified extension products, wherein said sequencing comprises use of a parallel sequencing platform; wherein said second adaptor sequence is ligated to one end but not both ends of said target nucleic acid fragment; and wherein said cancer gene comprises a somatic mutation.

34. The method of claim 16 , wherein said cancer gene comprises a somatic mutation.

35. The method of claim 16 , wherein said cancer gene comprises KRAS.

36. The method of claim 16 , further comprising performing targeted resequencing of said cancer gene in a plurality of samples.

37. The method of claim 16 , wherein said single-stranded DNA fragment is from a formalin-fixed paraffin-embedded sample.

38. The method of claim 16 , wherein said single-stranded DNA fragment comprises a target genomic region and a second adaptor sequence, and wherein said second adaptor sequence is different than said adaptor sequence of said target-specific primer-probes; wherein said sequencing comprises use of a parallel sequencing platform; wherein said second adaptor sequence is ligated to one end but not both ends of said single-stranded DNA fragment; and wherein said cancer gene comprises a somatic mutation.

39. The method of claim 1 , wherein said exon is larger than 500 bases.

40. The method of claim 16 , wherein said exon is larger than 500 bases.

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

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

Assignees

• Univ Leland Stanford Junior

Inventors

• Myllykangas Samuel
• Buenrostro Jason D
• Ji Hanlee P

Classifications

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title