Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis.
Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis.
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DOI：
10.1093/nargab/lqac045
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发表时间：
2022-06
影响因子：
4.6
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中科院分区：
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RNA分子中存在5-甲基胞嘧啶(M5C)已知数十年，其在调节RNA新陈代谢中的重要性已逐渐被认识到。尽管最近在对RNA M5C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，我们比较了四种RNA亚硫酸氢盐测序文库的构建方法，并实现了一种分析管道来评估M5C调用过程中的关键参数。我们发现，亚硫酸氢盐转化后的RNA碎片显著提高了产量，另外，额外的高温处理提高了亚硫酸氢盐的转化效率，特别是对定位于线粒体转录组的序列读出。使用唯一分子识别符(UMI)，我们观察到聚合酶链式反应有利于未甲基化模板的扩增。亚硫酸氢盐转化的碱基测序质量低是甲基化伪影的主要原因。此外，我们发现线粒体转录本经常对亚硫酸氢盐转换产生抵抗力，并且在线粒体mRNAs上找不到具有高置信度的p-M5C位点。综上所述，这项研究揭示了RNA亚硫酸氢盐测序数据中各种伪影的来源，并提供了一种改进的实验程序和分析方法。
The presence of 5-methylcytosine (m5C) 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 m5C 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 m5C 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.