Method for constructing next-generation sequencing library for detection of lowfrequency mutation and kit thereof

Abstract

The present invention provides a method for constructing a next-generation sequencing library for detecting low-frequency mutations, and a kit thereof. The constructing method comprises steps of obtaining blunt-end DNA fragments, obtaining DNA fragments with A-tail at the 3′ end, obtaining adapter-added DNA fragments using a specific nucleotide sequence and obtaining amplification products using a specific nucleotide sequence.

1 - 3 . (canceled) 
     
     
         4 . A kit for constructing a second-generation DNA sequencing library for detecting DNA low-frequency mutations, comprising:
 a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 1 and a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 2, or an annealing product thereof; and   a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 3.   
     
     
         5 . The kit according to  claim 4 , further comprising a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 4 and a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 5. 
     
     
         6 . A method for detecting DNA low-frequency mutations, comprising:
 step A: end-repairing DNA fragments to be sequenced in a sample containing low-frequency mutant DNA to obtain a blunt-end DNA fragments;   step B: A-tailing of a 3′ end to the blunt-end DNA fragments to obtain DNA fragments with an A-tail at the 3′ end;   step C: annealing single-stranded DNA having a nucleotide sequence of SEQ ID NO: 1 to single stranded DNA of SEQ ID NO: 2 thereby creating at least two adapters, adding the adapters to the DNA fragments with A-tail to the 3′ end to obtain a first adapter-added DNA fragment and a second adapter-added DNA fragment;   step D: binding a single-stranded DNA target primer having a nucleotide sequence of SEQ ID NO: 3 to one of the two adapter-added DNA fragments thereby creating a primer-adapter-added DNA fragment, subjecting the primer-adapter-added DNA fragment to PCR amplification to obtain amplification products; and   step E: conducting next-generation sequencing to the PCR amplification products and conducting bioinformatic analysis based on the sequencing result; wherein   the PCR amplification is conducted only once in said step D in this method.   
     
     
         7 . The method according to  claim 6 , wherein a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 4 and a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 5 are further used as PCR amplification primers in said step D. 
     
     
         8 . The method according to  claim 6 , wherein the amount of the DNA fragments in step A is 5 to 50 ng. 
     
     
         9 . A kit for detecting DNA low-frequency mutations, comprising:
 reagents for constructing a next-generation sequencing DNA library, and   reagents for computer sequencing a next-generation sequencing DNA library;   wherein the reagents for constructing a next generation sequencing DNA library comprises:   a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 1 and a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 2, or an annealing product thereof; and   a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 3.   
     
     
         10 . The kit according to  claim 9 , wherein the reagents for constructing a next-generation sequencing DNA library further comprises a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 4 and a single-stranded DNA having a nucleotide sequence such as shown in SEQ ID NO: 5.