Massive parallel sequencing of human whole mitochondrial genomes with Ion Torrent technology: an optimized workflow for Anthropological and Population Genetics studies

Investigation of human mitochondrial DNA variation patterns and phylogeny has been extensively used in Anthropological and Population Genetics studies and sequencing the whole mitochondrial genome is progressively becoming the gold standard. Among the currently available massive parallel sequencing technologies, Ion Torrent™ semiconductor sequencing represents a promising approach for such studies. Nevertheless, an experimental protocol conceived to enable the achievement of both as high as possible yield and of the most homogeneous sequence coverage through the whole mitochondrial genome is still not available. The present work was thus aimed at improving the overall performance of whole mitochondrial genomes Ion Torrent™ sequencing, with special focus on the capability to obtain robust coverage and highly reliable variants calling. For this purpose, a series of cost-effective modifications in standard laboratory workflows was fine-tuned to optimize them for medium- and large-scale population studies. A total of 54 human samples were thus subjected to sequencing of the whole mitochondrial genome with the Ion Personal Genome Machine™ System in four distinct experiments and using Ion 314 chips. Seven of the selected samples were also characterized by means of conventional Sanger sequencing for the sake of comparison. Obtained results demonstrated that the implemented optimizations had definitely improved sequencing outputs in terms of both variants calling efficiency and coverage uniformity, enabling to setup an effective and accurate protocol for whole mitochondrial genome sequencing and a considerable reduction in experimental time consumption and sequencing costs.

人类线粒体DNA（mitochondrial DNA）变异模式与系统发育的研究已广泛应用于人类学与群体遗传学领域，而全线粒体基因组测序也正逐步成为该领域的金标准。在当前可用的大规模并行测序（massive parallel sequencing）技术中，Ion Torrent™半导体测序技术为这类研究提供了一种极具潜力的方案。然而，目前仍缺乏一套能够同时实现尽可能高的测序产出与全线粒体基因组范围内最均匀的序列覆盖度的实验方案。因此，本研究旨在优化全线粒体基因组Ion Torrent™测序的整体性能，重点关注获得稳定覆盖度与高可靠性变异检出（variant calling）的能力。为此，本研究对标准实验室实验流程中的一系列低成本优化方案进行了微调，以适配中、大规模群体遗传学研究的需求。本研究共纳入54份人类样本，通过4组独立实验并使用Ion 314芯片，借助Ion Personal Genome Machine™系统完成了全线粒体基因组测序。为便于对照验证，其中7份入选样本同时采用传统Sanger测序进行了特征鉴定。研究结果表明，所实施的优化方案显著提升了测序产出在变异检出效率与覆盖均一度两方面的表现，成功建立了一套高效准确的全线粒体基因组测序实验方案，并大幅缩短了实验耗时、降低了测序成本。