Mitochondria whole genome sequencing method based on high-throughput sequencing

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 mean