Next-generation sequencing libraries
Provided herein is technology relating to next-generation sequencing and particularly, but not exclusively, to methods and compositions for preparing a next-generation sequencing library comprising short overlapping DNA fragments and using the library to sequence one or more target nucleic acids.
1. A method of generating a sequencing library, the method comprising:
a) amplifying a target nucleotide sequence using a primer comprising a target specific sequence and a universal sequence A to provide an amplicon, wherein the amplicon may be single stranded or double stranded;
b) ligating a first adaptor oligonucleotide comprising a universal sequence B to the amplicon to form an adaptor-amplicon; and
c) generating a ladder fragment library for use as a sequencing library, wherein said ladder fragment library comprises a plurality of nucleic acid fragments terminated by a 3′-O-propargyl nucleotide.
2. The method of claim 1 wherein the primer further comprises a barcode nucleotide sequence associated with the target nucleic acid.
3. The method of claim 2 wherein the barcode nucleotide sequence comprises 1 to 20 nucleotides.
4. The method of claim 1 wherein the first adaptor oligonucleotide is ligated to the 3′ end of the amplicon.
5. The method of claim 1 further comprising circularizing the adaptor-amplicon to form a circular template.
6. The method of claim 1 further comprising ligating a second adaptor oligonucleotide comprising a universal sequence C to the 3′ ends of the fragments of the ladder fragment library to generate a sequencing library.
7. The method of claim 6 comprising use of a click chemistry reaction to ligate the second adaptor oligonucleotide comprising a universal sequence C to the 3′ ends of the fragments of the ladder fragment library to generate the sequencing library.
8. The method of claim 1 wherein the first adaptor oligonucleotide comprises 10 to 80 nucleotides.
9. The method of claim 1 , wherein the nucleotide sequences of the fragments of the ladder fragment library correspond to overlapping nucleotide subsequences within the target nucleotide sequence and the nucleotide sequences of the fragments have 3′ ends corresponding to different nucleotides of the target nucleotide sequence.
10. The method of claim 1 wherein each nucleotide sequence of each fragment of the ladder fragment library comprises 15 to 1,000 nucleotides.
11. The method of claim 1 wherein generating said ladder fragment library comprises using a primer complementary to said universal sequence A.