Payne Brendan A I, Gardner Kristian, Coxhead Jonathan, Chinnery Patrick F
Mitochondrial Research Group, Institute of Genetic Medicine, Newcastle University, International Centre for Life, Central Parkway, Newcastle-upon-Tyne, NE1 3BZ, UK,
Methods Mol Biol. 2015;1264:59-66. doi: 10.1007/978-1-4939-2257-4_6.
Detecting and quantifying low-level variants in mitochondrial DNA (mtDNA) by deep resequencing can lead to important insights into the biology of mtDNA in health and disease. Massively parallel ("next-generation") sequencing is an attractive tool owing to the great depth and breadth of coverage. However, there are several important challenges to be considered when using this method, in particular: the avoidance of false discovery due to the unintended amplification of nuclear pseudogenes and the approach to delineating signal from noise at very great depths of coverage. Here we present methods for whole mtDNA genome deep sequencing (Illumina MiSeq) and short amplicon deep sequencing (Roche 454 GS-FLX).
通过深度重测序检测和定量线粒体DNA（mtDNA）中的低水平变异，可深入了解mtDNA在健康和疾病中的生物学特性。由于覆盖深度和广度大，大规模平行（“下一代”）测序是一种有吸引力的工具。然而，使用该方法时需考虑几个重要挑战，特别是：避免因核假基因的意外扩增导致的假阳性发现，以及在极深覆盖度下区分信号与噪声的方法。在此，我们介绍了用于全mtDNA基因组深度测序（Illumina MiSeq）和短扩增子深度测序（Roche 454 GS-FLX）的方法。
