High-throughput targeted library building method and application

Abstract

The invention discloses a high-throughput targeted library building method and application. The high-throughput targeted library building method comprises the following steps that a targeted capture probe and a PCR amplification primer are designed and synthesized based on a detected gene; a genome DNA of a sample is hybridized with the targeted capture probe overnight to obtain a captured targetfragment, and cyclizing, digesting, purifying and PCR amplification are carried out to obtain a constructed targeted sequencing library; and the targeted sequencing library is purified, the concentration and size of DNA fragments of the library are detected, second-generation sequencing is carried out, and the quality and sequencing information of the targeted sequencing library are analyzed according to a sequencing result. According to the high-throughput targeted library building method and application, hundreds of detection genes can be amplified in a round of PCR reaction, so that the defects that only a small number of gene loci can be detected by multiplex PCR library construction and experimental conditions are difficult to optimize are overcome; only one turn of PCR is performed,and random barcode are arranged on a probe framework, so that PCR bias is reduced, and base errors introduced by PCR and sequencing can be distinguished; and the library building step is simplified, and the reagent and time cost is reduced.

Description

technical field [0001] The invention relates to the technical field of sequencing detection, in particular to a method and application of high-throughput targeted library construction. Background technique [0002] Next-generation sequencing has been increasingly recognized and applied in molecular testing; it can be divided into targeted sequencing, whole-transcriptome sequencing and whole-genome sequencing. Targeted sequencing is an efficient and feasible method to obtain disease-related gene information, but because the key links are limited by existing methods, the cost is still relatively expensive, and there are shortcomings such as cumbersome operations and low sensitivity. Targeted library construction is a key link in gene-targeted sequencing. At present, domestic and foreign sequencing targeted library construction methods mainly use multiplex PCR or probe capture methods. It is difficult to explore the experimental conditions for multiplex PCR library constructio...

AI Technical Summary

Problems solved by technology

It is difficult to explore the experimental conditions for multiplex PCR library construction, and there are shortcomings such as the small number of detected genes, the inability to remove PCR bias, and the detection method's sensitivity is not high

The method of probe capture library construction requires the synthesis of special probes, which is expensive

These two technologies are expensive and cannot be applied quickly and flexibly according to actual needs

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