A high-throughput simplified genome sequencing library construction method

Abstract

The invention provides a method for constructing a high-flux simplified genome sequencing library. The method comprises the following steps: digesting genome DNA of different samples by using restriction enzymes, thereby obtaining blunt-end DNA fragments with phosphorylation modification at the 5' end; adding the base A at the 3' end, connecting the DNA fragments of different samples with 5' phosphorylation and 3' cohesive ends A with joints containing different bar code sequences, connecting the products PCR, purifying and mixing the amplification products, performing low-cycle PCR amplification, thereby obtaining a target fragment amplification product of which the ratio of the joints to joint dimers is greatly reduced; and separating, purifying, and forming the high-flux simplified genome sequencing library from the purified products. The method disclosed by the invention has the advantages that the flux is high, the cost is low, and the generated library is high in sequencing quality and has very wide application prospects in researches such as high-flux SNP detection and marker development, genetic map drawing and functional gene location.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a high-throughput simplified genome sequencing library. Background technique [0002] With the development and upgrading of next-generation sequencing technology, especially the Illumina sequencing platform, the research of population genetics has undergone revolutionary changes. Using next-generation sequencing, hundreds of thousands or even millions of SNPs information can be obtained, and more and more Multiple economically important animals and economic crops were located in candidate areas closely associated with important economic traits. At present, the next-generation sequencing library construction methods that can be used for population genetics research mainly include whole genome resequencing (WGR, Whole genome resequencing), RAD-Seq (Restriction-site-association DNA sequencing), GBS (Genotyping-by-Sequencing), etc. However, these methods have d...

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Problems solved by technology

At present, the next-generation sequencing library construction methods that can be used for population genetics research mainly include whole genome resequencing (WGR, Whole genome resequencing), RAD-Seq (Restriction-site-association DNA sequencing), GBS (Genotyping-by-Sequencing), etc. However, these methods have different problems. For example, WGR needs a high-quality reference genome sequence and the cost of building a library is high; although RAD-seq can be used for species without a reference genome, the amount of genomic DNA is large and the process is complicated. And the sequencing quality is poor, nearly half of the original data will be discarded due to sequencing errors; although the steps of GBS are relatively simple, it can only collect short fragments and the number of fragments is relatively small

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