Method for simultaneously detecting copy number and mutation of mtDNA based on NGS

Abstract

The invention discloses a method for simultaneously detecting the copy number and mutation of mtDNA based on NGS. A capture probe of mitochondrion DNA and 6 reference DNA fragments is prepared, the mitochondrion DNA and references in a whole-genome sequencing library are captured, sequencing data is analyzed by utilizing a bioinformatics technology, and simultaneous accurate detection of mitochondrial genome mutation and the copy number is realized. The method specifically comprises the following steps of 1, PCR amplification; 2, probe preparation; 3, capture sequencing; 4, bioinformatics analysis. Compared with the prior art, the method has the advantages that the low-frequency mutation and the copy number of the mitochondrion DNA can be accurately detected in the capture sequencing process, the DNA demand is reduced, precious DNA samples are saved, the experiment operation is simplified, and the detection cost is greatly reduced. Six nuclear DNA fragments are randomly selected as the references in the preparation process of the probe, the problem that capture sequencing data is used for calculating the copy number in the prior art and accordingly the accuracy is poor is solved, and the accuracy of related research results is improved.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for simultaneously detecting mtDNA copy number and mutation based on next-generation sequencing technology (NGS). Background technique [0002] Because the mitochondrial genome (mtDNA) has been exposed to high levels of reactive oxygen species for a long time, it lacks the protection of histones and the effective DNA damage repair system, and its mutation frequency is much higher than that of the nuclear genome. A large number of studies have shown that mtDNA variation is significantly related to the risk and prognosis of various diseases. By detecting the variation of free mtDNA in body fluid, it is expected to become a new technology for early diagnosis and monitoring of diseases. mtDNA variation mainly includes two forms of copy number variation and mutation. Therefore, the precise detection technology for mtDNA copy number and mutation has attracted the attenti...

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the above technical defects, provide a method for simultaneously detecting mtDNA copy number and mutation based on NGS, realize the simultaneous capture and sequencing of mtDNA and internal reference genes, and overcome the DNA requirements for separate detection of mtDNA mutation and copy number in the past Large volume, complex operation, high cost, etc., taking into account the characteristics of high sequencing depth requirements for mutation detection and small variation in internal reference sequencing depth for copy number detection, to provide an economical and effective technical solution for simultaneous detection of mtDNA mutations and copy number

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