Paired-end library construction method and method for sequencing genome by using library

Abstract

The invention relates to a paired-end library construction method and a method for sequencing a genome by using the library. The paired-end library construction method comprises the following steps of: performing plasmid extraction on clone in a constructed genome DNA library, fragmenting the extracted plasmid DNA, performing methyl protection on the restriction endonuclease site on the fragmented DNA, adding a hairclip type joint, performing enzyme cutting and cyclization to obtain circular DNA, then amplifying the genome library paired-end fragment in the circular DNA through a pair of composite primers to obtain a paired-end sequence of ultra-long span, and finally performing high flux sequencing. The paired-end sequence obtained by using the method can be applied to splicing of a new species genome sequence so as to further improve the splicing quality.

Description

Technical field [0001] The invention relates to a method for constructing a paired paired-end library and a method for genome sequencing using the library. Background technique [0002] The establishment and use of gene libraries is a development of recombinant DNA technology in the early 1970s. In order to isolate genes, especially those of eukaryotes, since 1974, organisms such as Escherichia coli, yeast, fruit flies, chickens, rabbits, mice, humans, soybeans and other organisms have been established successively, as well as the mitochondrial and chloroplast DNA of some organisms. Gene library. The establishment of gene library has brought the research of molecular genetics and genetic engineering into a new era. [0003] After the genomic DNA of an organism is partially digested with restriction enzymes, the digested fragments are inserted into the vector DNA molecules. The collection of all these vector molecules inserted with genomic DNA fragments will contain the entire gen...

AI Technical Summary

Problems solved by technology

Now different sequencing companies have launched different Paired-end library preparation methods, such as Roche, Illumina, and are widely used, but the span of their Paired-end library is within 20kb, mainly because the existing preparation methods first need to The extracted genomic DNA is interrupted to a certain size with HydrogenShear. The larger the fragment length, the lower the recovery rate, resulting in a larger initial genome amount; at the same time, the larger the fragment length, the worse the homogeneity after interruption, which will affect the later genome assembly. It has an impact; moreover, there is a step in the middle of library preparation that requires the fragment to be self-circularized. The larger the fragment, the more difficult it is to circularize, and the success rate is relatively lower.

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