Method for precisely determining mitochondrion whole genome sequence of eriocheir sinensis

Abstract

The invention relates to a method for precisely determining a mitochondrion whole genome sequence of eriocheir sinensis and the whole genome sequence obtained by using the method. The method comprisesthe following steps of (1) extracting the whole genome DNA in eriocheir sinensis muscle tissues, building a gene library, and performing sequencing by using a second generation sequencing technology;(2) screening a sequencing read segment by referring to the mitochondrion whole genome sequence of closely related species; eliminating nuclear DNA segments; then performing mitochondrion genome splicing; and performing prediction annotation on the protein-coding genes, rRNA and tRNA of the spliced mitochondrion genome; and (3) comparing the spliced mitochondrion genome sequence and the mitochondrion genome sequence of the closely related species; performing PCR amplification on a corresponding second generation sequencing sample according to a conservative region by referring to a mitochondrion whole genome sequence design primer of the closely related species; sequencing a PCR amplification product by using a first generation sequencing technology; and correcting the spliced mitochondrion genome sequence. The method has the advantages of high speed and high accuracy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for accurately measuring the whole mitochondrial genome sequence of the Chinese mitten crab and the whole mitochondrial genome sequence of the Chinese mitten crab measured by the method. Background technique [0002] Mitochondria has a double-membrane structure and unique DNA molecules. It is also the only semi-autonomous organelle with an "extrachromosomal" genome in animal cells. It also has the ability to transmit and express a complete genetic information system. In recent years, the mitochondrial genome has become a very characteristic molecular marker because of its unique properties, such as: small molecular weight, simple structure, maternal inheritance, no introns, high evolution rate, and no gene recombination. Due to the importance and particularity of mitochondrial structure, function and heredity, mitochondrial DNA has been widely used in phylogeneti...

AI Technical Summary

Problems solved by technology

The main feature of the first-generation sequencing technology is that the sequencing read length can reach 1000bp, and the accuracy is as high as 99.999%, but its disadvantages such as high sequencing cost and low throughput seriously affect its real large-scale application

Therefore, first-generation sequencing technology is not the most ideal sequencing method

Second-generation sequencing technology (also known as "high-throughput sequencing technology") has greatly reduced the cost of sequencing and greatly increased the speed of sequencing, but its application is limited by read fragments (200bp–500bp), and its accuracy is also low. Lower than the first-generation sequencing technology, the main reason is the substitution of bases

Compared with the previous two generations, the third-generation sequencing technology is characterized by single-molecule sequencing, which is fast and can read 10 dNTPs per second, but its sequencing error rate is relatively high, reaching 15%, which is almost the current Common faults of single-molecule sequencing technology

[0004] When sequencing the mitochondrial genome, ① if the first-generation sequencing technology is used, it will cause problems such as high sequencing cost and heavy workload, which will seriously affect its real large-scale application, so the first-generation sequencing technology is not ideal The sequencing method; ② If the second-generation sequencing technology (also known as "high-throughput sequencing technology") is used, the accuracy of its sequencing will be reduced, which will affect the final result

③Compared with the previous two generations, the third-generation sequencing technology also has the disadvantage of higher sequencing error rate

It can be seen that none of these sequencing technologies can ideally obtain the mitochondrial genome

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