Direct Capture, Amplification and Sequencing of Target DNA Using Immobilized Primers

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.

1 - 22 . (canceled) 
     
     
         23 . 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 from a tissue sample comprising a cancer cell, wherein the target nucleic acid fragment comprises (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.   
     
     
         24 . The method of  claim 23 , wherein said hybridization, said extension, and said amplification steps are performed directly inside a next-generation DNA sequencer. 
     
     
         25 . The method of  claim 23 , wherein said target-specific primer-probes are selectively hybridizable to said target genomic region of interest. 
     
     
         26 . The method of  claim 23 , further comprising sequencing said amplified extension products. 
     
     
         27 . The method of  claim 26 , wherein said sequencing comprises use of a parallel sequencing platform. 
     
     
         28 . The method of  claim 23 , wherein said amplification comprises bridge polymerase chain reaction (PCR). 
     
     
         29 . The method of  claim 23 , further comprising ligating said second adaptor sequence to said target nucleic acid fragment. 
     
     
         30 . The method of  claim 23 , wherein said cancer gene comprises a somatic mutation. 
     
     
         31 . The method of  claim 23 , wherein said cancer gene comprises KRAS. 
     
     
         32 . The method of  claim 23 , further comprising performing targeted resequencing of said cancer gene in a plurality of samples. 
     
     
         33 . The method of  claim 23 , wherein said tissue sample is from a formalin-fixed paraffin-embedded sample. 
     
     
         34 . A method for targeted sequencing, comprising:
 (a) hybridizing target specific primer-probes to a single-stranded DNA fragment from a tumor sample, wherein said target-specific primer-probes comprise an adaptor sequence and 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.   
     
     
         35 . The method of  claim 34 , 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. 
     
     
         36 . The method of  claim 34 , wherein said adaptor sequence comprises a sequencing platform-specific sequence. 
     
     
         37 . The method of  claim 34 , wherein said sequencing platform comprises a next-generation DNA sequencer. 
     
     
         38 . The method of  claim 34 , wherein said second adaptor sequence is ligated to one end but not both ends of said single-stranded DNA fragment. 
     
     
         39 . The method of  claim 34 , wherein said second adaptor sequence comprises a barcode sequence. 
     
     
         40 . The method of  claim 34 , wherein said cancer gene comprises a somatic mutation. 
     
     
         41 . The method of  claim 34 , wherein said cancer gene comprises KRAS. 
     
     
         42 . The method of  claim 34 , wherein said single-stranded DNA fragment is from a formalin-fixed paraffin-embedded sample.