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

• US 10,072,283 B2
• Filed: 06/09/2014
• Issued: 09/11/2018
• Est. Priority Date: 09/24/2010
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Myllykangas, Samuel, Buenrostro, Jason D., Ji, Hanlee P.
• Primary Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US14/300,048
• Publication Number:
  US 20150017635A1
• Time in Patent Office: 1,555 Days
• Field of Search: None
• 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 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

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.

Claims

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.

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.

39. The method of claim 1, 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.

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.

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.

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

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