Mitochondria whole genome sequencing method based on high-throughput sequencing

Abstract

The invention discloses a simple, economical and accurate mitochondria whole genome sequencing barcode amplification sequencing method based on second generation sequencing. Two groups of matched primers are designed, wherein the first group of primers is composed of 61 pairs of target fragment primer sequences and primer joint sequences which are overlaid and cover the mitochondria whole genome.The second group of primers is composed of positive and negative label primers which comprise the joint sequences and are matched two by two for marking samples of different sources. By matching the two pairs of primers in use, two pairs of the primers are added in a primary PCR reaction for amplification to obtain amplicons which have marks, are moderate in length and comprise mitochondria wholegenome. The amplicons are mixed and used for establishing a library and sequencing directly to achieve multi-sample mixed sequencing. Sequencing data identifies mark sequences at the head and tail ends at the same time by means of applying a bioinformatics assembling technology to different sequences of different sources to obtain all mitochondria genome sequence information of all samples. By employing the method, the sequencing experiment steps are simplified, the sequencing cost is lowered greatly, and the detection rate of low-frequency mutation is reduced, thereby providing probability for human mitochondria whole genome associated researches.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for sequencing the whole genome of mitochondrial DNA. Background technique [0002] Mitochondria are important organelles for metabolism and energy conversion in organisms. Human mitochondrial DNA (mtDNA) is a double-stranded closed circular molecule composed of 16571bp deoxyribonucleic acid, and the protein encoded by it is an important component of the respiratory chain. It plays an important role in forensic science, anthropology and genetics research. mtDNA has very high variability at the population level, and specific mtDNA variants affect mitochondrial function. This change may not only cause abnormalities in oxidative phosphorylation and energy metabolism, but also cause changes in related pathways such as apoptosis, which play an important role in the occurrence and development of aging and many complex diseases. It is hypothesized that the same...

AI Technical Summary

Problems solved by technology

However, the two-step PCR method will increase a lot of workload in practical applications, especially for sample amplification.

Recently, it was proposed that the primer sequence with barcode can be directly connected to the target sequence primer to reduce the workload of PCR amplification. However, if this method is applied to the amplification and sequencing of more fragments, the cost of primers will be reduced. And the consumption of barcode sequence is greatly increased

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