Self-priming and replicating hairpin adaptor for constructing NGS library, and method for constructing NGS library using same
View Patent ↗The present invention pertains to: a self-priming and replicating hairpin adaptor for constructing an NGS (next generation sequencing) library; and a method for constructing an NGS library using the same. Specifically, the present invention pertains to: a self-priming and replicating hairpin adaptor for constructing an NGS library, the adaptor containing a long single-stranded first oligonucleotide that comprises a nucleotide sequence of SEQ ID NO: 4 and has a hairpin structure, and a short single-stranded second oligonucleotide that comprises a nucleotide sequence of SEQ ID NO: 5; a method for constructing an NGS library using said adaptor; and an NGS library construction kit including said adaptor.
1 . A self-priming and replicating hairpin adaptor for next-generation sequencing (NGS) library preparation comprising: a long single-stranded first oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 and having a hairpin structure; and a short single-stranded second oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 5.
2 . The self-priming and replicating hairpin adaptor of claim 1 , wherein polymerase binds to the first oligonucleotide, and the hairpin structure is maintained even during an extension reaction.
3 . The self-priming and replicating hairpin adaptor of claim 1 , wherein the 5′ end of the second oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 is not phosphorylated so as not to be ligated to the 3′ end of the first oligonucleotide.
4 . A method for next-generation sequencing (NGS) preparation comprising steps of:
(1) fragmenting genomic DNA to be analyzed;
(2) ligating the self-priming and replicating hairpin adaptor of claim 1 to an end of the fragmented genomic DNA to be analyzed;
(3) adding polymerase to a reaction solution containing the fragmented genomic DNA to which the self-priming and replicating hairpin adaptor has been ligated in step (2), followed by an extension reaction to obtain a first reaction product;
(4) ligating a universal adaptor for NGS to the first reaction product;
(5) adding polymerase to a reaction solution containing the first reaction product to which the universal adaptor for NGS has been ligated in step (4), followed by an extension reaction to obtain a second reaction product; and
(6) purifying the second reaction product.
5 . The method of claim 4 , wherein the fragmented genomic DNA to be analyzed and the self-priming and replicating hairpin adaptor, which are used in step (2), are mixed with each other at a molar ratio of 1 (genomic DNA):15 (adaptor) to 1:25.
6 . The method of claim 4 , further comprising, before ligating the self-priming and replicating hairpin adaptor in step (2) and before ligating the universal adaptor for NGS in step (4), a step of adding adenosine to the 3′ end of the DNA (A-tailing) after end repair of the DNA.
7 . The method of claim 4 , wherein the first reaction product in step (4) is an amplification product comprising the nucleotide sequence of the self-priming and replicating hairpin adaptor and having an extended hairpin structure.
8 . The method of claim 4 , wherein the second reaction product in step (5) is in the form of linear duplex DNA formed by amplification in the extension reaction.
9 . The method of claim 4 , wherein each of the extension reaction in step (3) and the extension reaction in step (5) is performed once.
10 . A kit for DNA library preparation comprising the self-priming and replicating hairpin adaptor for next-generation sequencing (NGS) library preparation according to claim 1 .