A method for accurately determining the whole mitochondrial genome sequence of Chinese mitten crab

Abstract

The invention relates to a method for accurately determining the whole mitochondrial genome sequence of Chinese mitten crab and the whole genome sequence obtained by the method. The method comprises the steps of: (1) extracting the whole genome DNA in the muscle tissue of the Chinese mitten crab, constructing a gene library, and performing sequencing by using second-generation sequencing technology; (2) performing sequencing reads with reference to the whole genome sequence of the mitochondria of a close relative species Screening, removing nuclear DNA fragments, and then splicing the mitochondrial genome, and then predicting and annotating the coding protein genes, rRNA and tRNA of the spliced ​​mitochondrial genome; (3) combining the assembled mitochondrial genome sequence and the mitochondrial genome sequence of closely related species Comparison, according to the conserved region, and then refer to the mitochondrial genome sequence of the related species to design primers, perform PCR amplification on the corresponding second-generation sequencing sample, use the first-generation sequencing technology to sequence the PCR amplification product, and correct the spliced Mitochondrial genome sequence. This method has the advantages of fast and accurate.

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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