A cost-efficient method for sequencing the human whole mitochondrial genome utilizing multiplex PCR-based next generation sequencing

Abstract

The mitochondrial genome of human is organized as a circular loop of double-stranded DNA with 16569 bp in length. Mitochondrial DNA (mtDNA) is extensively used as a biomarker in human evolution and population genetics due to the characteristics of maternal inheritance, multiple copies in cells, high mutational rates, high heteroplasmy levels and so on. Next-generation sequencing (NGS) has become a rapid and efficient approach to sequence mtDNA recently, while library-building is complex and expensive. Multiplex PCR has the advantage of high sensitivity and saving time and effort. Therefore, this technique was used to amplify whole complete mitochondrial genomes in Chinese in the present study. The whole mitochondrial genome was covered by 73 overlapped short amplicons, which was subsequently sequenced on an Illumina HiSeq X Ten instrument. The mean per-mtDNA-base read depth was higher than 20000×. 100% mitochondrial genome coverage was obtained across all the samples with a depth of coverage threshold of 100×. The data of high throughout sequencing was qualified for mutation analysis. The present sequencing method based on multiplex PCR leaves out the process of library-building. This sequencing methodology that we have developed is efficiency, economic and has a broad application prospect in the research of complex genetic disorders.