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