Department of Data Analysis and Mathematical Modelling, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
HIV Cure Research Center, Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University and Ghent University Hospital, Ghent, Belgium.
PLoS One. 2018 Jun 14;13(6):e0199091. doi: 10.1371/journal.pone.0199091. eCollection 2018.
DNA methylation is one of the most important epigenetic modifications in the regulation of gene transcription. The current gold standard to study this modification is bisulfite sequencing. Although multiple commercial bisulfite treatment kits provide good conversion efficiencies, DNA loss and especially DNA fragmentation remain troublesome. This hampers DNA methylation profiling of long DNA sequences. Here, we explored the performance of twelve commercial bisulfite kits by an in-depth comparison of DNA fragmentation using gel electrophoresis, qPCR and digital PCR, DNA recovery by spectroscopic measurements and digital PCR and conversion efficiency by next generation sequencing. The results show a clear performance difference between the bisulfite kits, and depending on the specific goal of the study, the most appropriate kit might differ. Moreover, we demonstrated that digital PCR is a valuable method to monitor both DNA fragmentation as well as DNA recovery after bisulfite treatment.
DNA 甲基化是基因转录调控中最重要的表观遗传修饰之一。目前研究这种修饰的金标准是亚硫酸氢盐测序。虽然有多种商业亚硫酸氢盐处理试剂盒提供了良好的转化率，但 DNA 丢失，尤其是 DNA 片段化仍然是个麻烦。这阻碍了长 DNA 序列的 DNA 甲基化分析。在这里，我们通过凝胶电泳、qPCR 和数字 PCR 比较了 DNA 片段化、分光光度法和数字 PCR 测量的 DNA 回收以及下一代测序的转化率，深入比较了 12 种商业亚硫酸氢盐试剂盒的性能。结果表明，亚硫酸氢盐试剂盒之间存在明显的性能差异，具体取决于研究的特定目标，最合适的试剂盒可能不同。此外，我们证明了数字 PCR 是一种监测亚硫酸氢盐处理后 DNA 片段化和 DNA 回收的有价值的方法。
