Lemsara Amina, Dieterich Christoph, Naarmann-de Vries Isabel S
Klaus Tschira Institute for Integrative Computational Cardiology, University Heidelberg, Heidelberg, Germany.
Department of Internal Medicine III (Cardiology, Angiology, and Pneumology), University Hospital Heidelberg, Heidelberg, Germany.
Methods Mol Biol. 2023;2624:241-260. doi: 10.1007/978-1-0716-2962-8_16.
RNA modifications exist in all kingdom of life. Several different types of base or ribose modifications are now summarized under the term "epitranscriptome." With the advent of high-throughput sequencing technologies, much progress has been made in understanding RNA modification biology and how these modifications can influence many aspects of RNA life. The most widespread internal modification on mRNA is m6A, which has been implicated in physiological processes as well as disease pathogenesis. Here, we provide a workflow for the mapping of m6A sites using Nanopore direct RNA sequencing data. Our strategy employs pairwise comparison of basecalling error profiles with JACUSA2. We outline a general strategy for RNA modification detection on mRNA and describe two specific use cases on m6A detection in detail. Use case 1: a sample of interest with modifications (e.g., "wild-type" sample) is compared to a sample lacking a specific modification type (e.g., "knockout" sample, here METTL3-KO) or Use case 2: a sample of interest with modifications is compared to a sample lacking all modifications (e.g., in vitro transcribed cDNA). We provide a detailed protocol on experimental and computational aspects. Extensive online material provides a snakemake pipeline to identify m6A positions in mRNA and to validate the results against a miCLIP-derived m6A reference set. The general strategy is flexible and can be easily adapted by users in different application scenarios.
RNA修饰存在于所有生命王国中。目前，几种不同类型的碱基或核糖修饰被统称为“表观转录组”。随着高通量测序技术的出现，在理解RNA修饰生物学以及这些修饰如何影响RNA生命的许多方面都取得了很大进展。mRNA上最普遍的内部修饰是N6-甲基腺苷（m6A），它与生理过程以及疾病发病机制有关。在这里，我们提供了一种使用纳米孔直接RNA测序数据定位m6A位点的工作流程。我们的策略采用JACUSA2对碱基识别错误图谱进行成对比较。我们概述了mRNA上RNA修饰检测的一般策略，并详细描述了m6A检测的两个具体用例。用例1：将具有修饰的感兴趣样本（例如“野生型”样本）与缺乏特定修饰类型的样本（例如“敲除”样本，此处为METTL3基因敲除样本）进行比较；或者用例2：将具有修饰的感兴趣样本与缺乏所有修饰的样本（例如体外转录的cDNA）进行比较。我们提供了关于实验和计算方面的详细方案。丰富的在线材料提供了一个Snakemake管道，用于识别mRNA中的m6A位置，并根据miCLIP衍生的m6A参考集验证结果。该一般策略具有灵活性，用户可以在不同的应用场景中轻松调整。
