PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.
PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.
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DOI：
10.1371/journal.pone.0139253
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
LaFramboise T
中科院分区：
文献类型：
--
作者：
Gould MP;Bosworth CM;McMahon S;Grandhi S;Grimberg BT;LaFramboise T
测序技术的最新进展使得能够准确地检测线粒体序列变异，即使是那些异质体位点的低丰度变异也是如此。通过丰富线粒体(相对于核)DNA的样本，可以节省相当大的测序成本。核DNA(NDNA)含量的减少也有助于避免核线粒体序列(Numts)导致的假阳性变体。我们使用两种不同的方法从人类细胞系和血液成分中分离完整的线粒体细胞器：磁珠结合方案和差异离心法。DNA被提取出来，并通过酶消化进一步浓缩为线粒体DNA(MtDNA)。仅需1 ng纯化的DNA即可进行文库制备和下一代序列(NGS)分析。以mtDNA(与nDNA)含量为指标，通过实时定量聚合酶链式反应和NGS读数分析测量，对各种浓缩方法进行评估和比较。在所考察的各种方法中，最优的方法是差速离心分离，然后进行核酸外切酶消化。这一策略在血液和细胞系中产生了35%的线粒体DNA读数，相当于基线的数百倍。该策略还避免了错误的变异调用，正如我们所展示的，这种错误的变异调用可能是由目前浓缩程序中的标准远程聚合酶链式反应方法引起的。这一优化程序允许mtDNA浓缩以实现高效和准确的大规模并行测序，从而能够从具有少量起始材料的样品中进行NGS。这将通过增加可能被多路复用的样本数量来降低成本，最终促进更好地了解线粒体相关疾病的努力。
Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.