Q-RRBS: a quantitative reduced representation bisulfite sequencing method for single-cell methylome analyses

Reduced representation bisulfite sequencing (RRBS) is a powerful method of DNA methylome profiling that can be applied to single cells. However, no previous report has described how PCR-based duplication-induced artifacts affect the accuracy of this method when measuring DNA methylation levels. For quantifying the effects of duplication-induced artifacts on methylome profiling when using ultra-trace amounts of starting material, we developed a novel method, namely quantitative RRBS (Q-RRBS), in which PCR-induced duplication is excluded through the use of unique molecular identifiers (UMIs). By performing Q-RRBS on varying amounts of starting material, we determined that duplication-induced artifacts were more severe when small quantities of the starting material were used. However, through using the UMIs, we successfully eliminated these artifacts. In addition, Q-RRBS could accurately detect allele-specific methylation in absence of allele-specific genetic variants. Our results demonstrate that Q-RRBS is an optimal strategy for DNA methylation profiling of single cells or samples containing ultra-trace amounts of cells.

简化代表性亚硫酸氢盐测序（Reduced representation bisulfite sequencing, RRBS）是一种强大的DNA甲基化组分析技术，可应用于单细胞样本。然而，此前尚无研究阐明基于PCR的复制诱导伪影在DNA甲基化水平检测中对该技术准确性的影响。为量化超微量起始样本条件下，复制诱导伪影对甲基化组分析的影响，我们开发了一种全新方法——定量简化代表性亚硫酸氢盐测序（quantitative RRBS, Q-RRBS），该方法通过引入唯一分子标识符（unique molecular identifiers, UMIs）来排除PCR诱导的复制伪影。通过对不同起始量样本开展Q-RRBS实验，我们发现当起始样本量较小时，复制诱导的伪影问题更为显著；而借助唯一分子标识符，我们成功消除了此类伪影。此外，Q-RRBS可在无等位基因特异性遗传变异的情况下，精准检测等位基因特异性甲基化。本研究结果证实，Q-RRBS是单细胞或含超微量细胞样本的DNA甲基化组分析的最优策略。