A simple method for sequencing the whole human mitochondrial genome directly from samples and its application to genetic testing
A simple method for sequencing the whole human mitochondrial genome directly from samples and its application to genetic testing
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DOI：
10.1038/s41598-019-53449-y
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发表时间：
2019-11-22
影响因子：
4.6
通讯作者：
Tanaka, Tomoaki
中科院分区：
文献类型：
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作者：
Yao, Yue;Nishimura, Motoi;Tanaka, Tomoaki
下一代测序（NGS）是一项革命性的基因组分析测序技术。然而，线粒体DNA （mtDNA）测序的预处理方法仍然很复杂，需要开发一种可靠的预处理方法。在这里，我们开发了一种简单易行的预处理方法，基于等温滚动循环mtDNA扩增，使用市售试剂。使用纳升血浆和从血液或组织样本中提取的总DNA 25 ng，成功地进行了mtDNA的等温扩增。在mtDNA扩增之前，有必要用外切酶V处理提取的总DNA，但不需要处理血浆。由扩增的mtDNA生成的NGS文库提供了整个人类线粒体基因组的测序覆盖范围。此外，测序结果成功地检测到患者样本中的异质性，与先前独立的Sanger测序分析相匹配的称为突变和变体。此外，在健康志愿者中检测到一种新的单核苷酸变异。使用患者非常小的样本成功分析mtDNA可能在临床医学中很有价值，因为它可以通过减少采样相关的组织损伤来减少患者的不适。总之，本文描述的简单方便的预处理方法可能促进未来基于ngs的临床和法医mtDNA检测的发展。
Next-generation sequencing (NGS) is a revolutionary sequencing technology for analyzing genomes. However, preprocessing methods for mitochondrial DNA (mtDNA) sequencing remain complex, and it is required to develop an authenticated preprocessing method. Here, we developed a simple and easy preprocessing method based on isothermal rolling circle mtDNA amplification using commercially available reagents. Isothermal amplification of mtDNA was successfully performed using both nanoliter quantities of plasma directly and 25 ng of total DNA extracted from blood or tissue samples. Prior to mtDNA amplification, it was necessary to treat the extracted total DNA with Exonuclease V, but it was not required to treat plasma. The NGS libraries generated from the amplified mtDNA provided sequencing coverage of the entire human mitochondrial genome. Furthermore, the sequencing results successfully detected heteroplasmy in patient samples, with called mutations and variants matching those from previous, independent, Sanger sequencing analysis. Additionally, a novel single nucleotide variant was detected in a healthy volunteer. The successful analysis of mtDNA using very small samples from patients is likely to be valuable in clinical medicine, as it could reduce patient discomfort by reducing sampling-associated damage to tissues. Overall, the simple and convenient preprocessing method described herein may facilitate the future development of NGS-based clinical and forensic mtDNA tests.