A double-enzyme digestion simplified genome next-generation sequencing library construction method and supporting kit

Abstract

The present invention provides a simplified genome next-generation sequencing library construction method and kit based on double-enzyme digestion. The present invention addresses the shortcomings of existing double-enzyme-digestion simplified genome sequencing library construction methods, expands the scope of double-enzyme-digestion combinations, and reduces the number of simplified Genomic library construction is overly dependent on expensive instruments, which simplifies the library construction process, reduces the cost of library construction and improves sequencing efficiency. At the same time, the technology is simple, flexible and easier to be mastered by researchers and can be implemented in ordinary molecular laboratories. . It is especially suitable for micro or medium-scale laboratories that need to conduct SNP molecular marker development, genetic map construction, population genetics research and phylogenetic biology research on a large number of species with incomplete reference genomes. The invention has good practical application value and application prospect in the fields of agricultural molecular breeding, conservation biology and evolutionary biology.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a simplified genome next-generation sequencing library based on double enzyme digestion and a matching kit for constructing the next-generation sequencing library. Background technique [0002] Restriction-site Associated DNA (RAD) sequencing technology, that is, RAD-seq technology is a simplified genome sequencing technology based on genome-wide restriction sites developed on the basis of next-generation sequencing ( Baird N.J., et al, 2008). This technology uses a restriction endonuclease to single-enzyme digest the genome, combined with physical methods to break it up to generate DNA fragments of a certain size and construct a sequencing library, so that high-throughput sequences near the restriction site can be achieved sequencing. Since the RAD marker is a DNA fragment near the genome-wide specific restriction site, it can represent the se...

AI Technical Summary

Problems solved by technology

[0005] Although ddRAD simplifies the library construction process to a certain extent compared to RAD, it still includes 11 steps, so the experiment takes a long time

At the same time, ddRAD performs double enzyme digestion on genomic DNA by combining a rare enzyme with a common enzyme. This combination is based on the consideration of the number of fragments produced by enzyme digestion, but it limits the choice of enzymes.

In addition, the experimental process uses magnetic beads to purify DNA and DNA fragment automatic recovery equipment to select fragments. The magnetic frame and DNA fragment automatic recovery instrument Pippin-Prep required for magnetic bead purification are relatively expensive test consumables and instruments. Ordinary molecules Labs are difficult to configure

In addition, the single-stranded oligonucleotides for the synthesis of P1 and P2 linkers require high-performance liquid chromatography (HPLC) purification and terminal phosphorylation, and the cost of synthesizing P1 and P2 linkers alone will be unaffordable for ordinary laboratories

Further, the lengths of the 48 barcode sequences provided by the ddRAD technical process are exactly the same, which will cause a serious imbalance in the bases of the restriction site, which will lead to a decrease in the quality of the sequencing at this position, and it is impossible to judge whether the library digestion is complete or not based on the quality value. asterisk activity

A large amount of time consumption and relatively high database construction costs seriously restrict the wide application of ddRAD

Therefore, it is still necessary to improve the existing ddRAD simplified genome sequencing library construction method to overcome the shortcomings of the existing method, such as limited range of enzyme selection, complex use of instruments, cumbersome process, high cost and low quality of base sequencing at the restriction site. , improve sequencing efficiency and enable library construction to be implemented smoothly in small and medium-sized laboratories with 5-10 people and insufficient funds, without relying on large regional instrument centers or sequencing centers

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