Method and kit for constructing next-generation sequencing library

Abstract

The invention discloses a method and a kit for constructing a next-generation sequencing library in the field of nucleic acid sequencing, and the method comprises the following steps: step 1, extracting genome DNA needing to be sequenced, including but not limited to DNA of various tumor tissues, or free DNA of peripheral blood or DNA of various body fluids, such as but not limited to DNA in hydrothorax, urine, saliva and the like, and step 2, designing a primer sequence containing a specific target sequence primer, a sample marker sequence (index) and a sequencing primer, and synthesizing the primer is artificially; step 3, mixing the specific primer with DNA to be detected, and carrying out PCR amplification; step 4, purifying a PCR product; step 5, sequencing on a machine. According to the method and the kid for constructing the next-generation sequencing library, the library construction process is simplified, the library construction cost is reduced, the detection sensitivity is improved, the sequencing data volume required by detection is also reduced, false positive is effectively removed, the target DNA fragment enrichment efficiency is improved, and sequencing data waste is reduced.

Description

technical field [0001] The invention relates to the field of nucleic acid sequencing, in particular to a method and a kit for constructing a next-generation sequencing library. Background technique [0002] Gene mutation refers to the change of base pair composition or sequence in gene structure. In pathogen detection, tumor mutation gene detection, and fetal cell-free DNA detection in pregnant women’s plasma, it is necessary to find a small number of mutant genes in a large number of normal gene sequences. At this time, next-generation sequencing is characterized by its high-throughput sequencing. It can exert excellent characteristics and detect a small amount (less than 1%) of mutated genes in samples at a cost much lower than that of generation sequencing (Sanger sequencing), such as free DNA (ctDNA) with tumor characteristics in the plasma of cancer patients, cancer There is a low proportion of subclonal mutations in tissue samples (such as FFPE) and fetal cell-free DN...

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Problems solved by technology

[0004] The purpose of the present invention is to provide a method and a kit for constructing a next-generation sequencing library to solve the problem that the loss of template DNA in the above-mentioned background technology is very large, which is very unfavorable for the detection of low-frequency gene mutations, and the accuracy of detection is more difficult to guarantee. The problem of increasing sequencing costs

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