一种基于多重PCR的人类线粒体基因组高通量测序方法

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

人类线粒体基因组DNA(mtDNA)是一个16569 bp的双链闭合环状DNA分子,具有母系遗传、多拷贝、高异质性及高变异率等特点,是研究人类遗传和进化上广泛使用的分子标记.近几年,高通量测序技术的出现,使得在短时间内准确测定mtDNA序列成为可能;但目前常用的高通量测序建库方法操作复杂、研究费用相对较高.基于多重PCR扩增的测序方法具有高效率、高灵敏度、低成本的特点,因而适用于大规模线粒体基因组的变异检测分析.利用73个相互重叠的扩增子通过多重PCR方法来扩增中国人的线粒体全基因组,在扩增片段两端连接特定的接头序列,然后在IlluminaHiSeq X Ten平台上进行高通量测序.对获得的测序数据分析发现,mtDNA每个位点的测序深度均达到2000×以上;当测序深度为100×时,所有样本的序列覆盖度都达到100%;数据质量适用于后续的变异检测分析.利用本研究建立的基于多重PCR的二代测序方法无需片段化即可直接上机测序,在复杂遗传病的研究中有着广泛的应用前景.

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 20000x. 100% mitochondrial genome coverage was obtained across all the samples with a depth of coverage threshold of 100x. 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.