Next-generation sequencing libraries

Abstract

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.

We claim: 
     
         1 . A kit for sequencing a target nucleic acid, the kit comprising:
 a) a nucleic acid ladder fragment library wherein said nucleic acid ladder fragment library comprises a plurality of nucleic acid fragments terminated by a 3′-O-propargyl nucleotide; and   b) an adaptor oligonucleotide.   
     
     
         2 . The kit of  claim 1 , wherein said 3′-O-propargyl nucleotide comprises a first reactive group and said adaptor oligonucleotide comprises a second reactive group that is capable of being linked to said first reactive group by click chemistry. 
     
     
         3 . The kit of  claim 1 , wherein said nucleic acid fragment ladder comprises a plurality of nucleic acids having 3′ ends that differ by less than 20 nucleotides. 
     
     
         4 . The kit of  claim 1 , further comprising a copper-based click chemistry catalyst reagent. 
     
     
         5 . The kit of  claim 1 , further comprising a computer readable medium comprising instructions to instruct a computer to assemble short overlapping nucleotide sequences and to produce a consensus sequence. 
     
     
         6 . The kit of  claim 5  wherein:
 i) each said short overlapping nucleotide sequence comprises less than 100 bases; 
 ii) said short overlapping nucleotide sequences are tiled over a target nucleic acid comprising at least 100 bases; and 
 iii) said short overlapping nucleotide sequences are offset from one another by 1-20 bases. 
 
     
     
         7 . The kit of  claim 1 , further comprising a polymerase. 
     
     
         8 . The kit of  claim 1 , further comprising a second adaptor oligonucleotide. 
     
     
         9 . The kit of  claim 1 , further comprising one or more compositions comprising a nucleotide or a mixture of nucleotides. 
     
     
         10 . The kit of  claim 1 , further comprising a ligase. 
     
     
         11 . The kit of  claim 1 , further comprising a sequencing primer.