Oxford Nanopore Direct RNA Sequencing datasets for detecting rRNA modifications in the Brassica oleracea mitoribosome

Oxford Nanopore Direct RNA Sequencing (DRS) was applied for the detection of rRNA modifications in the Brassica oleracea mitoribosome. A comparison between native rRNA transcripts and in vitro transcribed (IVT) rRNA transcripts that were devoid of any modification indicated systematic base-calling errors and/or variations in current intensities that led to the prediction of the modified nucleotides (Begik et al., 2018). For Nanopore (DRS) library preparation, custom reverse transcription adapters (RTAs) containing Deeplexicon multiplexing barcodes (BC1, BC2 or BC3) were designed for sequence-specific ligation to the 3’-ends of B. oleracea mitochondrial rRNAs. Basecalling and demultiplexing of ONT direct RNA sequencing data were performed by Guppy and Deeplexicon, respectively. For the analysis and visualization of current intensities, a full description is available in the associated publication. The Eventalign_18S.R Rscript was used to make the nanopore 18S signal analysis The Eventalign_26S.R Rscript was used to make the nanopore 26S signal analysis The raw data for the scripts are in the following folders: - For 26S: 26S_Native.barcode01 and 26S_IVT.barcode03- For 18S: 18S_Native.barcode01 and 18S_IVT.barcode02 These folders contain the read alignment files output from minimap2 (in .bam format) and the eventalign files generated by the f5c software (in tsv format). The FASTA folder contains the mitochondrial 18S and 26S rRNA gene reference sequence in fasta format牛津纳米孔直接RNA测序（Oxford Nanopore Direct RNA Sequencing，DRS）用于检测芸薹（Brassica oleracea）线粒体核糖体的rRNA修饰。将天然rRNA转录本与无任何修饰的体外转录（in vitro transcribed，IVT）rRNA转录本进行比对后，发现存在系统性碱基识别错误和/或电流强度变异，据此可预测修饰核苷酸（Begik等，2018）。 在纳米孔DRS文库构建流程中，设计了带有Deeplexicon多重条形码（BC1、BC2或BC3）的定制反转录接头（custom reverse transcription adapters，RTAs），用于与芸薹线粒体rRNA的3'端进行序列特异性连接。 ONT直接RNA测序数据的碱基识别与解多重（demultiplexing）处理分别由Guppy和Deeplexicon完成。 关于电流强度的分析与可视化的完整说明，可参见相关已发表研究论文。 使用Eventalign_18S.R R脚本完成纳米孔18S信号分析。 使用Eventalign_26S.R R脚本完成纳米孔26S信号分析。 上述脚本的原始数据存放于以下文件夹： - 针对26S：26S_Native.barcode01与26S_IVT.barcode03 - 针对18S：18S_Native.barcode01与18S_IVT.barcode02 这些文件夹包含由minimap2输出的读段比对文件（格式为.bam），以及由f5c软件生成的eventalign文件（格式为.tsv）。 FASTA文件夹中存放了线粒体18S和26S rRNA基因的参考序列，格式为fasta。