<b>R2Dtool: Integration and visualization of</b><b> isoform-</b><b>resolved</b><b> RNA features</b>

Long-read RNA sequencing enables the mapping of RNA modifications, structures, and protein-interaction sites at the resolution of individual transcript isoforms. To understand the functions of these RNA features, it is critical to analyze them in the context of transcriptomic and genomic annotations, such as open reading frames and splice junctions. To enable this, we have developed R2Dtool, a bioinformatics tool that integrates transcript-mapped information with transcript and genome annotations, allowing for the isoform-resolved analytics and graphical representation of RNA features in their genomic context. We illustrate R2Dtool’s capability to integrate and expedite RNA feature analysis using epitranscriptomics data. m6A modification calls were generated from nanopore direct RNA sequencing of HeLa transcriptomic RNA (SQK-RNA004 kit). DRACH-context m6A basecalling was performed with Dorado v0.5.3 (https://github.com/nanoporetech/dorado) and the methylation calls were processed with ModKit v0.2.6 (https://github.com/nanoporetech/modkit), before analysis by R2Dtool. R2Dtool software and analysis steps are available at https://github.com/comprna/R2Dtool.长读长RNA测序（long-read RNA sequencing）可在单个转录本异构体分辨率下，实现RNA修饰、RNA结构及蛋白质互作位点的定位分析。为解析此类RNA特征的生物学功能，需结合转录组与基因组注释信息（如开放阅读框、剪接接头）对其开展分析，这一步至关重要。为此，我们开发了生物信息学工具R2Dtool，该工具可整合转录本比对信息与转录组、基因组注释信息，能够在基因组背景下实现RNA特征的异构体分辨率分析与可视化呈现。我们利用表观转录组学（epitranscriptomics）数据，展示了R2Dtool整合并加速RNA特征分析的能力。本研究采用SQK-RNA004试剂盒，对HeLa细胞的转录组RNA进行纳米孔直接RNA测序，以此生成m6A修饰位点调用结果。随后使用Dorado v0.5.3（https://github.com/nanoporetech/dorado）完成DRACH基序背景下的m6A碱基识别，并通过ModKit v0.2.6（https://github.com/nanoporetech/modkit）处理甲基化位点调用结果，最终交由R2Dtool开展后续分析。R2Dtool软件及完整分析流程可于https://github.com/comprna/R2Dtool获取。