Johnson Zachary, Xu Xiguang, Pacholec Christina, Xie Hehuang
Epigenomics and Computational Biology Lab, Fralin Life Sciences Institute, Virginia Tech, Blacksburg, VA 24061, USA.
NAR Genom Bioinform. 2022 Jun 3;4(2):lqac045. doi: 10.1093/nargab/lqac045. eCollection 2022 Jun.
The presence of 5-methylcytosine (mC) in RNA molecules has been known for decades and its importance in regulating RNA metabolism has gradually become appreciated. Despite recent advances made in the functional and mechanistic understanding of RNA mC modifications, the detection and quantification of methylated RNA remains a challenge. In this study, we compared four library construction procedures for RNA bisulfite sequencing and implemented an analytical pipeline to assess the key parameters in the process of mC calling. We found that RNA fragmentation after bisulfite conversion increased the yield significantly, and an additional high temperature treatment improved bisulfite conversion efficiency especially for sequence reads mapped to the mitochondrial transcriptome. Using Unique Molecular Identifiers (UMIs), we observed that PCR favors the amplification of unmethylated templates. The low sequencing quality of bisulfite-converted bases is a major contributor to the methylation artifacts. In addition, we found that mitochondrial transcripts are frequently resistant to bisulfite conversion and no p-m5C sites with high confidence could be identified on mitochondrial mRNAs. Taken together, this study reveals the various sources of artifacts in RNA bisulfite sequencing data and provides an improved experimental procedure together with analytical methodology.
RNA分子中5-甲基胞嘧啶（mC）的存在已为人所知数十年，其在调节RNA代谢中的重要性也逐渐得到认可。尽管最近在对RNA mC修饰的功能和机制理解方面取得了进展，但甲基化RNA的检测和定量仍然是一个挑战。在本研究中，我们比较了用于RNA亚硫酸氢盐测序的四种文库构建程序，并实施了一个分析流程来评估mC检测过程中的关键参数。我们发现，亚硫酸氢盐转化后的RNA片段化显著提高了产量，额外的高温处理提高了亚硫酸氢盐转化效率，特别是对于映射到线粒体转录组中的序列读数。使用独特分子标识符（UMIs），我们观察到PCR有利于未甲基化模板的扩增。亚硫酸氢盐转化碱基的低测序质量是甲基化假象的主要原因。此外，我们发现线粒体转录本经常对亚硫酸氢盐转化有抗性，并且在线粒体mRNA上无法确定具有高可信度的p-m5C位点。综上所述，本研究揭示了RNA亚硫酸氢盐测序数据中假象的各种来源，并提供了一种改进的实验程序以及分析方法。
