Primer for amplicon sequencing and two-step PCR database building method

Abstract

The invention provides a primer for amplicon sequencing and a two-step PCR database building method. The method comprises the following steps of: 1) extracting genomic DNA of a species sample to be tested; 2) respectively connecting universal primer sequences to 5' ends of forward and reverse primers of the multiplex PCR, and taking the genomic DNA as a template to carry out a first round of multiplex PCR to obtain a PCR product with universal primer sequences at both ends; 3) purifying the product of the first round of PCR; 4) respectively connecting primer sequences matched with the first round of PCR universal primer sequences at both ends of the sequencing label sequence to obtain a PCR product carrying the label sequence, and using the PCR product as a template to carry out a second round of PCR; and 5) purifying the PCR product, and sequencing on the machine after passing the quality inspection. The invention adopts the two-step PCR method to build a database, so that the amplicon sequencing cost is reduced, the detection cost is reduced by more than 50%, and the quality index of the amplicon is effectively improved. That is to say, the detection rate of the site and the datauniformity are improved, and the method has extremely high economic value.

Description

technical field [0001] The invention relates to molecular biology and bioinformatics, in particular to a primer for amplicon sequencing and a two-step PCR library construction method. Background technique [0002] Amplicon sequencing is a highly targeted targeted region sequencing method, which is used to analyze gene variation in a specific genomic region. Ultra-high depth sequencing of PCR products (amplicons) can effectively identify and analyze variation characteristics. Amplicon sequencing uses the designed primer sequences to target the target region, and then performs high-throughput sequencing (NGS), analyzes the sequencing results, and obtains corresponding information. [0003] Since the existing amplicon sequencing technology mainly uses end-joining method and adapters to build a library, the steps are cumbersome and the material cost of each sample is higher than 300 yuan, so there is an urgent need to develop a lower-cost sequencing method. Contents of the inv...

AI Technical Summary

Problems solved by technology

[0003] Since the existing amplicon sequencing technology mainly uses end-joining method to build a library, the steps are cumbersome and the material cost of each sample is higher than 300 yuan, so there is an urgent need to develop a lower-cost sequencing method

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