Multipurpose DNA segment enrichment method used for next generation sequencing

Abstract

The invention discloses a preparation method of a DNA sequencing template. The preparation method comprises following step: (1) DNA molecules to be sequenced are subjected to fragmentation, end terminal repair and supplement, A addition on 3' end, and terminal connection; (2) enrichment of target DNA segments is realized via three times of semi-nested PCR amplification, wherein a primer used in the former two times comprises a specific primer and an universal primer, a primer used in the third time is composed of two universal primers, and the PCR amplification is multiplex-PCR reaction in the former two times. Compared with Illumina GA sample pretreatment processes, the preparation method is lack of a step of sample treatment, so that time is shortened, cost is reduced, and demanded amount of the initial DNA sample is reduced; in addition, the most significant is that reduction of cycling times and application of Phusion high fidelity DNA polymerase are capable of reducing mutation caused by PCR, so that processing on obtained data becomes relatively simple and clear, and when the preparation method is used for a plurality of re-sequencing experiments, demand on sequencing depth is reduced.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for preparing a DNA sequencing template, in particular to a method for enriching target DNA fragments for next-generation sequencing analysis. Background technique [0002] Next Generation Sequencing (NGS) is the general term for a series of emerging sequencing technologies in recent years. They are all based on the principle of Sequencing by Synthesis, with high throughput, short time and huge data volume. Its characteristics have been widely used in genome (re)sequencing, transcriptome sequencing, single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) development, etc. powerful tool. [0003] Among various next-generation sequencing technologies, the Genome Analyzer (GA) developed by Illumina has become one of the most widely used technologies due to its advantages of relatively low cost, large throughput, and simple pre-processing. one. [0004] The D...

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Problems solved by technology

[0009] The above three methods are all completed before the Illumina GA sample pretreatment, and the techniques involved in aqueous phase hybridization and solid phase hybridization are relatively complicated and costly (especially the acquisition of solid phase supports for DNA microarrays requires a large amount of synthesis specific sequence), the amount of starting genomic DNA required is large, and the obtained target fragments still need to be enriched after processing (usually also by PCR)

Although PCR is a method with good selectivity and low technical requirements, base mutations will occur during amplification. Although the use of high-fidelity polymerases can improve the frequency of mutations, but with the amplification cycle As the number increases, the probability of base mutation will increase

Since there are 18 cycles of PCR amplification in the Illumina GA sample pretreatment, the base mutations introduced during the PCR reaction before the Illumina GA sample pretreatment will be amplified in the subsequent Illumina GA sample pretreatment, and the sequence obtained The sequencing quality of mutated bases is not significantly different from that of unmutated bases, and cannot be distinguished, which will be harmful when detecting point mutations or single nucleotide polymorphisms (SNP). The results are very noisy, causing too many false positive results

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