Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), National Research Council, Bari, Italy.
Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy.
Nat Protoc. 2020 Mar;15(3):1098-1131. doi: 10.1038/s41596-019-0279-7. Epub 2020 Jan 29.
RNA editing is a widespread post-transcriptional mechanism able to modify transcripts through insertions/deletions or base substitutions. It is prominent in mammals, in which millions of adenosines are deaminated to inosines by members of the ADAR family of enzymes. A-to-I RNA editing has a plethora of biological functions, but its detection in large-scale transcriptome datasets is still an unsolved computational task. To this aim, we developed REDItools, the first software package devoted to the RNA editing profiling in RNA-sequencing (RNAseq) data. It has been successfully used in human transcriptomes, proving the tissue and cell type specificity of RNA editing as well as its pervasive nature. Outcomes from large-scale REDItools analyses on human RNAseq data have been collected in our specialized REDIportal database, containing more than 4.5 million events. Here we describe in detail two bioinformatic procedures based on our computational resources, REDItools and REDIportal. In the first procedure, we outline a workflow to detect RNA editing in the human cell line NA12878, for which transcriptome and whole genome data are available. In the second procedure, we show how to identify dysregulated editing at specific recoding sites in post-mortem brain samples of Huntington disease donors. On a 64-bit computer running Linux with ≥32 GB of random-access memory (RAM), both procedures should take ~76 h, using 4 to 24 cores. Our protocols have been designed to investigate RNA editing in different organisms with available transcriptomic and/or genomic reads. Scripts to complete both procedures and a docker image are available at https://github.com/BioinfoUNIBA/REDItools.
RNA 编辑是一种广泛存在的转录后机制，能够通过插入/缺失或碱基替换来修饰转录本。它在哺乳动物中尤为突出，在哺乳动物中，ADAR 酶家族的成员将数百万个腺苷脱氨酶转化为肌苷。A-to-I RNA 编辑具有丰富的生物学功能，但在大规模转录组数据集检测仍然是一个未解决的计算任务。为此，我们开发了 REDItools，这是第一个专门用于 RNA 测序 (RNAseq) 数据中 RNA 编辑分析的软件包。它已成功应用于人类转录组，证明了 RNA 编辑的组织和细胞类型特异性及其普遍性。从人类 RNAseq 数据的大规模 REDItools 分析中收集的结果已包含在我们的专用 REDIportal 数据库中，该数据库包含超过 450 万个事件。在这里，我们详细描述了基于我们的计算资源 REDItools 和 REDIportal 的两个生物信息学程序。在第一个程序中，我们概述了一种在可获得转录组和全基因组数据的人类细胞系 NA12878 中检测 RNA 编辑的工作流程。在第二个程序中，我们展示了如何在亨廷顿病供体的死后脑组织样本中识别特定重编码位点的失调编辑。在运行 Linux 的 64 位计算机上，具有 ≥32GB 的随机存取内存 (RAM)，这两个程序都应该使用 4 到 24 个核，大约需要 76 小时。我们的方案旨在使用具有可用转录组和/或基因组读数的不同生物体来研究 RNA 编辑。完成这两个程序的脚本和一个 docker 映像可在 https://github.com/BioinfoUNIBA/REDItools 上获得。
