Methods of Generating Gene Specific Libraries
The invention provides compositions and methods for generating a target enriched, sequencing ready library for resequencing at least one target region of interest from a nucleic acid containing sample.
1 .- 15 . (canceled)
16 . A method of enriching a library for target nucleic acid regions of interest, the method comprising:
(a) contacting a library of DNA molecules comprising a subpopulation of nucleic acid target insert sequences of interest flanked by a first primer binding region and a second primer binding region within a larger population of nucleic acid insert sequences flanked by the first primer binding region and the second primer binding region with a set of capture probes, the set of capture probes comprising a plurality of capture oligonucleotides, each comprising a first target sequence-specific binding region and a second capture reagent binding region, under conditions that allow binding between the capture oligonucleotides and the nucleic acid target regions of interest, to form a mixture comprising a plurality of complexes between target regions of interest and capture probes;
(b) contacting the mixture of step (a) with a capture reagent and separating the capture reagent bound complex from the mixture; and
(c) eluting the target regions of interest flanked by the first primer binding region and the second primer binding region from the capture reagent bound complex.
17 . The method of claim 16 , wherein the second capture reagent binding region directly binds to the capture reagent.
18 . The method of claim 16 , wherein the second capture reagent binding region binds to an adaptor capture oligonucleotide comprising a region that binds to the capture reagent; wherein the method further comprises contacting the mixture of step (a) with a plurality of adaptor capture oligonucleotides.
19 . The method of claim 16 , wherein step (a) is carried out in a solution comprising from 100 mM to 2 M NaCl.
20 . The method of claim 16 , further comprising washing the separated capture reagent bound complex with a wash solution comprising less than 10 mM NaCl prior to step (c).
21 . The method of claim 17 , wherein the wash solution further comprises from 15% to 30% formamide.
22 . The method of claim 13 , wherein the set of capture probes comprises a plurality of capture oligonucleotides, each capture probe comprising a first target-specific binding region that is at least 95% identical to at least a portion of the sense or antisense strand of the exons in at least 5 different genes.
23 . The method of claim 16 , wherein the set of capture probes comprises a plurality of capture oligonucleotides, each comprising a first target-specific binding region that is at least 95% identical to at least a portion of the sense or antisense strand of the exons in at least 70 different genes.
24 . The method of claim 16 , further comprising amplifying the eluted target regions of interest flanked by the first primer binding region and the second primer binding region with a forward PCR primer and a reverse PCR primer that bind to the first and second primer binding regions to generate a library that is once-enriched for target regions of interest.
25 . The method of claim 24 , further comprising:
(d) contacting the library that is once-enriched for target regions of interest with the capture probe pool under conditions that allow binding between the capture oligonucleotides and the nucleic acid target regions of interest, to form a plurality of complexes between target regions of interest and capture probes;
(e) contacting the mixture of step (d) with a capture reagent and separating the capture reagent bound complex from the mixture; and
(f) eluting the target regions of interest flanked by the first primer binding region and the second primer binding region from the capture reagent bound complex.
26 . The method of claim 25 further comprising amplifying the eluted target regions of interest flanked by the first primer binding region and the second primer binding region with a forward PCR primer and a reverse PCR primer that bind to the first and second primer binding regions to generate a library that is twice-enriched for target regions of interest.
27 . A method of generating a target enriched, sequencing ready library for resequencing at least one target region of interest from a nucleic acid containing sample, the method comprising:
(a) providing a library comprising fragmented nucleic acid molecules flanked by a first primer binding region and a second primer binding region; and
(b) enriching the library for target sequences with a set of capture probes comprising a plurality of capture oligonucleotides, each comprising a first target-specific binding region and a second capture reagent binding region, thereby generating an enriched sequencing ready library for resequencing at least one target region of interest.
28 . The method of claim 27 , further comprising PCR amplifying the enriched library with PCR primers that bind to the first primer binding region and the second primer binding region to generate an amplified product.
29 . The method of claim 27 , wherein at least one of the first stem-loop linker or the second stem-loop linker oligonucleotides comprises a molecular bar code.
30 . The method of claim 27 , further comprising sequencing at least a portion of the enriched library to determine the sequence of the regions of interest.
31 . The method of claim 27 , wherein the library is generated from nucleic acids obtained from a human subject.
32 . The method of claim 28 , further comprising sequencing at least a portion of the enriched library to determine the sequence of the regions of interest.