Tytgat Olivier, Tang Mao-Xing, van Snippenberg Willem, Boel Annekatrien, Guggilla Ramesh Reddy, Gansemans Yannick, Van Herp Michiel, Symoens Sofie, Trypsteen Wim, Deforce Dieter, Heindryckx Björn, Coucke Paul, De Spiegelaere Ward, Van Nieuwerburgh Filip
Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
Department of Life Science Technologies, Imec, Leuven, Belgium.
Clin Chem. 2021 Jul 6;67(7):968-976. doi: 10.1093/clinchem/hvab021.
The quantification of mitochondrial DNA heteroplasmy for the diagnosis of mitochondrial disease or after mitochondrial donation, is performed mainly using next-generation sequencing strategies (NGS). Digital PCR (dPCR) has the potential to offer an accurate alternative for mutation load quantification.
We assessed the mutation load of 23 low-input human samples at the m.11778 locus, which is associated with Leber's hereditary optic neuropathy (LHON) using 2 droplet digital PCR platforms (Stilla Naica and Bio-Rad QX200) and the standard NGS strategy. Assay validation was performed by analyzing a titration series with mutation loads ranging from 50% to 0.01%.
A good concordance in mutation rates was observed between both dPCR techniques and NGS. dPCR established a distinctly lower level of background noise compared to NGS. Minor alleles with mutation loads lower than 1% could still be detected, with standard deviations of the technical replicates varying between 0.07% and 0.44% mutation load. Although no significant systematic bias was observed when comparing dPCR and NGS, a minor proportional bias was detected. A slight overestimation of the minor allele was observed for the NGS data, most probably due to amplification and sequencing errors in the NGS workflow.
dPCR has proven to be an accurate tool for the quantification of mitochondrial heteroplasmy, even for samples harboring a low mutation load (<1%). In addition, this alternative technique holds multiple benefits compared to NGS (e.g., less hands-on time, more straightforward data-analysis, and a lower up-front capital investment).
线粒体DNA异质性的定量分析主要用于线粒体疾病的诊断或线粒体捐赠后，目前主要采用下一代测序策略（NGS）。数字PCR（dPCR）有潜力为突变负荷定量提供一种准确的替代方法。
我们使用2种液滴数字PCR平台（Stilla Naica和Bio-Rad QX200）以及标准的NGS策略，评估了23份低输入量人类样本在与Leber遗传性视神经病变（LHON）相关的m.11778位点的突变负荷。通过分析突变负荷范围从50%到0.01%的滴定系列进行分析方法验证。
两种dPCR技术与NGS之间在突变率上具有良好的一致性。与NGS相比，dPCR的背景噪声水平明显更低。仍可检测到突变负荷低于1%的次要等位基因，技术重复的标准差在突变负荷的0.07%至0.44%之间变化。虽然在比较dPCR和NGS时未观察到显著的系统偏差，但检测到了轻微的比例偏差。在NGS数据中观察到次要等位基因略有高估，很可能是由于NGS工作流程中的扩增和测序错误。
dPCR已被证明是一种用于定量线粒体异质性的准确工具，即使对于低突变负荷（<1%）的样本也是如此。此外，与NGS相比，这种替代技术具有多种优势（例如，操作时间更少、数据分析更直接以及前期资本投资更低）。
