Zambelli Filippo, Vancampenhout Kim, Daneels Dorien, Brown Daniel, Mertens Joke, Van Dooren Sonia, Caljon Ben, Gianaroli Luca, Sermon Karen, Voet Thierry, Seneca Sara, Spits Claudia
Research Group Reproduction and Genetics, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
S.I.S.Me.R. Reproductive Medicine Unit, Bologna, Italy.
Eur J Hum Genet. 2017 Nov;25(11):1229-1236. doi: 10.1038/ejhg.2017.129. Epub 2017 Aug 23.
Massive parallel sequencing (MPS) can accurately quantify mitochondrial DNA (mtDNA) single nucleotide variants (SNVs), but no MPS methods are currently validated to simultaneously and accurately establish the breakpoints and frequency of large deletions at low heteroplasmic loads. Here we present the thorough validation of an MPS protocol to quantify the load of very low frequency, large mtDNA deletions in bulk DNA and single cells, along with SNV calling by standard methods. We used a set of well-characterized DNA samples, DNA mixes and single cells to thoroughly control the study. We developed a custom script for the detection of mtDNA rearrangements that proved to be more accurate in detecting and quantifying deletions than pre-existing tools. We also show that PCR conditions and primersets must be carefully chosen to avoid biases in the retrieved variants and an increase in background noise, and established a lower detection limit of 0.5% heteroplasmic load for large deletions, and 1.5 and 2% for SNVs, for bulk DNA and single cells, respectively. Finally, the analysis of different single cells provided novel insights into mtDNA cellular mosaicism.
大规模平行测序（MPS）能够准确地对线粒体DNA（mtDNA）单核苷酸变异（SNV）进行定量分析，但目前尚无MPS方法能够在低异质性负荷情况下同时准确地确定大片段缺失的断点和频率。在此，我们展示了一种MPS方案的全面验证，该方案用于定量分析大量DNA和单细胞中极低频率的大片段mtDNA缺失负荷，并通过标准方法进行SNV检测。我们使用了一组特征明确的DNA样本、DNA混合物和单细胞来全面控制该研究。我们开发了一个用于检测mtDNA重排的定制脚本，该脚本在检测和定量缺失方面比现有工具更为准确。我们还表明，必须谨慎选择PCR条件和引物组，以避免在检索到的变异中出现偏差以及背景噪声增加，并分别确定了大量DNA和单细胞中大片段缺失的异质性负荷检测下限为0.5%，SNV的检测下限分别为1.5%和2%。最后，对不同单细胞的分析为mtDNA细胞镶嵌现象提供了新的见解。
