Patent ID: 9650628
Date: 2017-05-16
CPC Classifications: C12N,C12Q

Claim:
1. A method for enriching for a nucleic acid sequence of interest in a sample comprising nucleic acids, the method comprising: (a) fragmenting nucleic acids in the sample comprising the nucleic acids, thereby generating nucleic acid fragments, wherein the nucleic acid fragments comprise the nucleic acid sequence of interest; (b) ligating a first adaptor sequence to a 5′ end of the nucleic acid fragments, thereby generating nucleic acid fragments ligated to the first adaptor sequence; (c) purifying the nucleic acid fragments ligated to the first adaptor sequence; (d) annealing one or more oligonucleotides in solution to the nucleic acid sequence of interest in the purified nucleic acid fragments ligated to the first adaptor sequence, wherein the one or more oligonucleotides in solution comprise a 3′ portion with at least 10 bases that are complementary to the nucleic acid sequence of interest and a 5′ tail, wherein the 5′ tail comprises a second adaptor sequence non-complementary to the sequence of interest; (e) extending the one or more oligonucleotides annealed to the nucleic acid sequence of interest in the nucleic acid fragments ligated to the first adaptor sequence with a polymerase in a reaction mixture, wherein the reaction mixture does not comprise a primer that anneals to sequence complementary to the first adaptor sequence, thereby generating one or more oligonucleotide extension products comprising sequence complementary to the ligated first adaptor sequence at a first end, sequence complementary to the nucleic acid sequence of interest, and the second adaptor sequence at a second end; (f) amplifying the one or more oligonucleotide extension products using a first primer that anneals to the complement of the first adaptor sequence and a second primer that anneals to a complement of the second adaptor sequence to enrich for the nucleic acid sequence of interest, wherein products of the amplifying comprise a 3′ end with sequence complementary to a sequence on a surface, thereby providing an enriched nucleic acid sequence of interest; (g) annealing a strand of the products of the amplifying to the sequence on the surface using the 3′ end with sequence complementary to the sequence on the surface; and (h) sequencing the enriched nucleic acid sequence of interest on a massively parallel sequencing platform.