一种基于线粒体分离纯化技术的新型线粒体DNA深度测序方法

A novel deep sequencing method for whole mitochondrial genome based on mitochondrial isolation and purification

目的 开发并评估一种基于线粒体分离纯化技术的线粒体DNA(mtDNA)深度测序方法。方法 探索从细胞中分离mtDNA的最适条件;基于该方法对外周血单个核细胞(PBMC)样本进行mtDNA深度测序,通过测序片段(reads)中mtDNA reads占比评估测序文库中mtDNA的纯度,通过Sanger测序验证该方法所检出的mtDNA变异的准确性。结果 采用含低浓度NP40和不含Tween-20的裂解液裂解细胞获得的mtDNA纯度高,几乎不含细胞核基因组。在6例PBMC样本中应用该方法检测结果显示,mtDNA reads占比为87.8%~92.8%,且检测出多个mtDNA变异。对其中1个异质性变异和3个同质性变异进行Sanger测序验证,二者结果相一致。结论 本研究开发的基于线粒体分离纯化技术的mtDNA深度测序方法可应用于PBMC样本,能准确且灵敏地检出线粒体基因组的同质性和异质性变异。

Objective To develop and evaluate a novel deep sequencing method for mitochondrial DNA(mtDNA)based on mitochondrial isolation and purification.Methods The optimal conditions for isolating mtDNA from cells were explored.Then,mtDNA from peripheral blood mononuclear cells(PBMC)was deeply sequenced.The purity of mtDNA in the sequencing library was evaluated by the proportion of mtDNA reads in the sequencing fragments(reads).The accuracy of mtDNA mutations detected by the established method was verified by Sanger sequencing.Results The mtDNA obtained by using the lysate containing low concentration of NP40 and without Tween-20 had high purity and almost no nuclear genome.The established method was used to detect six samples of PBMC,and the results showed that the proportion of mtDNA reads ranged from 87.8%to 92.8%,and that multiple mtDNA mutations were detected.One heterogenetic variation and three homogenetic variations were verified by Sanger sequencing,and the results were consistent.Conclusion The established deep sequencing method for mtDNA based on mitochondrial isolation and purification can be applied to PBMC samples and accurately and sensitively detect the heterogeneity and homogeneity of mitochondrial genome.