Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.
Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.
Methods. 2019 Mar 1;156:121-127. doi: 10.1016/j.ymeth.2018.10.013. Epub 2018 Oct 24.
RNA cytosine-5 methylation (mC) 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 mC marks at nucleotide resolution. Targeted bisulfite sequencing allows mC detection at single base resolution, by combining the use of tailored primers with bisulfite treatment. A number of computational tools currently exist to analyse mC marks in DNA bisulfite sequencing. However, these methods are not directly applicable to the analysis of RNA mC 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 mC 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.
RNA 胞嘧啶-5 甲基化 (mC) 已成为一种关键的转录后修饰标记，它在结构调节、应激信号和蛋白质翻译调控中发挥多种作用。亚硫酸氢盐测序目前是检测核苷酸分辨率 mC 标记最准确、最可靠的方法。靶向亚硫酸氢盐测序通过结合使用定制引物和亚硫酸氢盐处理，可以实现 mC 在单碱基分辨率上的检测。目前存在许多用于分析 DNA 亚硫酸氢盐测序中 mC 标记的计算工具。然而，这些方法不能直接应用于 RNA mC 标记的分析，因为 DNA 分析侧重于 CpG 甲基化，并且 RNA 中的人为未转化和错误扩增会掩盖实际的甲基化信号。我们描述了一个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。该管道可直接应用于 Illumina MiSeq（或等效）测序数据集，并通过优化的映射参数和针对去除人工制品的定制过滤器的应用来定义。我们提供了应用此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 中的 mC 标记以及在人成纤维细胞中的 28S rRNA 中的 mC 标记的示例。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 扩增子中的胞嘧啶-5 甲基化提供了一个准确、快速和用户友好的框架。
