RNA cytosine-5 methylation (m5C) has emerged as a key epitranscriptomic mark, which fulfills multiple roles in structural modulation, stress signaling and the regulation of protein translation. Bisulfite sequencing is currently the most accurate and reliable method to detect m5C marks at nucleotide resolution. Targeted bisulfite sequencing allows m5C detection at single base resolution, by combining the use of tailored primers with bisulfite treatment. A number of computational tools currently exist to analyse m5C marks in DNA bisulfite sequencing. However, these methods are not directly applicable to the analysis of RNA m5C marks, because DNA analysis focuses on CpG methylation, and because artifactual unconversion and misamplification in RNA can obscure actual methylation signals. We describe a pipeline designed specifically for RNA cytosine-5 methylation analysis in targeted bisulfite sequencing experiments. The pipeline is directly applicable to Illumina MiSeq (or equivalent) sequencing datasets using a web interface (https://bisamp.dkfz.de), and is defined by optimized mapping parameters and the application of tailored filters for the removal of artifacts. We provide examples for the application of this pipeline in the unambiguous detection of m5C marks in tRNAs from mouse embryonic stem cells and neuron-differentiated stem cells as well as in 28S rRNA from human fibroblasts. Finally, we also discuss the adaptability of BisAMP to the analysis of DNA methylation. Our pipeline provides an accurate, fast and user-friendly framework for the analysis of cytosine-5 methylation in amplicons from bisulfite-treated RNA.

中文翻译：

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BisAMP：用于靶向 RNA 胞嘧啶-5 甲基化分析的基于网络的管道

RNA 胞嘧啶 5 甲基化 (m5C) 已成为关键的表观转录组标记，它在结构调节、应激信号传导和蛋白质翻译调节中发挥多种作用。亚硫酸氢盐测序是目前以核苷酸分辨率检测 m5C 标记的最准确、最可靠的方法。通过将定制引物的使用与亚硫酸氢盐处理相结合，靶向亚硫酸氢盐测序允许以单碱基分辨率检测 m5C。目前存在许多计算工具来分析 DNA 亚硫酸氢盐测序中的 m5C 标记。然而，这些方法并不直接适用于 RNA m5C 标记的分析，因为 DNA 分析侧重于 CpG 甲基化，并且因为 RNA 中的人为未转换和错误放大会掩盖实际的甲基化信号。我们描述了专门为靶向亚硫酸氢盐测序实验中的 RNA 胞嘧啶-5 甲基化分析而设计的管道。该管道直接适用于使用 Web 界面 (https://bisamp.dkfz.de) 的 Illumina MiSeq（或等效）测序数据集，并由优化的映射参数和用于去除伪影的定制过滤器的应用定义。我们提供了该管道在明确检测来自小鼠胚胎干细胞和神经元分化干细胞的 tRNA 以及来自人类成纤维细胞的 28S rRNA 中的 m5C 标记的应用示例。最后，我们还讨论了 BisAMP 对 DNA 甲基化分析的适应性。我们的管道为分析亚硫酸氢盐处理的 RNA 扩增子中胞嘧啶 5 甲基化提供了准确、快速和用户友好的框架。