Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation
The present invention provides methods, compositions and kits for targeted nucleic acid sequence enrichment in a nucleic acid sample and for high efficiency nucleic acid library generation for next generation sequencing (NGS). Specifically, the methods, compositions and kits provided herein are useful for the production and capture of amplification-ready, target-specific and strand-specific regions of interest from nucleic acid samples containing complex DNA.
1. A method for sequencing an enriched nucleic acid sequence of interest, the method comprising:
a) annealing one or more oligonucleotides in solution in a reaction mixture to the nucleic acid sequence of interest in a nucleic acid fragment, wherein the reaction mixture comprises a plurality of nucleic acid fragments, wherein the nucleic acid fragment comprising the nucleic acid sequence of interest comprises a first adaptor sequence, wherein the one or more oligonucleotides comprise a 3′ portion with at least 10 bases designed to be complementary to the nucleic acid sequence of interest and a 5′ tail portion comprising a second adaptor sequence that is non-complementary to the nucleic acid sequence of interest;
b) extending the one or more oligonucleotides annealed to the nucleic acid sequence of interest in the nucleic acid fragment comprising the first adaptor sequence with a polymerase, in the reaction mixture, thereby generating one or more oligonucleotide extension products comprising sequence complementary to the 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;
c) amplifying the one or more oligonucleotide extension products, in the reaction mixture, using a first primer that anneals to the complement of the first adaptor sequence and a second primer that anneals at its 3′ end to a complement of the second adaptor sequence, thereby enriching the nucleic acid sequence of interest, by generating amplified products comprising the enriched nucleic acid sequence of interest; and
d) sequencing the amplified products comprising the enriched nucleic acid sequence of interest on a massively parallel sequencing platform.
2. The method of claim 1 , wherein the nucleic acid sequence of interest comprises genomic DNA.
3. The method of claim 1 , wherein the nucleic acid sequence of interest comprises cDNA.
4. The method of claim 1 , wherein the nucleic acid sequence of interest comprises RNA.
5. The method of claim 1 , wherein the first adaptor sequence and the second adaptor sequence are distinct from each other.
6. The method of claim 1 , wherein the plurality of nucleic acid fragments comprises the first adaptor sequence, and wherein the first adaptor sequence is common to the plurality of nucleic acid fragments.
7. The method of claim 1 , wherein the one or more oligonucleotides comprises more than one oligonucleotide, wherein the second adaptor sequence is common to the more than one oligonucleotide.
8. The method of claim 1 , wherein the first adaptor sequence and/or the second adaptor sequence comprise a barcode sequence.
9. The method of claim 1 , further comprising denaturing double-stranded nucleic acid fragments prior to step a).
10. The method of claim 1 , wherein the polymerase is a DNA polymerase.
11. The method of claim 1 , wherein the sequencing comprises the use of four labeled reversible terminators.
12. The method of claim 1 , wherein the sequencing comprises semiconductor sequencing.
13. The method of claim 1 , wherein the sequencing comprises nanopore sequencing.
14. The method of claim 1 , wherein each of the plurality of nucleic acid fragments comprises the first adaptor sequence at a 5′ end.
15. The method of claim 1 , further comprising fragmenting nucleic acids prior to step a), thereby generating the plurality of nucleic acid fragments.
16. The method of claim 1 , further comprising ligating the first adaptor sequence to a nucleic acid fragment in the plurality of nucleic acid fragments prior to step a), thereby generating the nucleic acid fragment comprising the first adaptor sequence.
17. The method of claim 1 , wherein the amplified products comprise a 3′ end with sequence complementary to a sequence on a surface.
18. The method of claim 17 , further comprising 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 prior to step d).
19. The method of claim 17 , wherein the surface is a surface of a flow cell.