R2Dtool: Integration and visualization of isoform-resolved RNA features

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.