Background: Direct-sequencing technologies, such as Oxford Nanopore's, are delivering long RNA reads with great efficacy and convenience. These technologies afford an ability to detect post-transcriptional modifications at a single-molecule resolution, promising new insights into the functional roles of RNA. However, realizing this potential requires new tools to analyze and explore this type of data.
Result: Here, we present Sequoia, a visual analytics tool that allows users to interactively explore nanopore sequences. Sequoia combines a Python-based backend with a multi-view visualization interface, enabling users to import raw nanopore sequencing data in a Fast5 format, cluster sequences based on electric-current similarities, and drill-down onto signals to identify properties of interest. We demonstrate the application of Sequoia by generating and analyzing ~ 500k reads from direct RNA sequencing data of human HeLa cell line. We focus on comparing signal features from m6A and m5C RNA modifications as the first step towards building automated classifiers. We show how, through iterative visual exploration and tuning of dimensionality reduction parameters, we can separate modified RNA sequences from their unmodified counterparts. We also document new, qualitative signal signatures that characterize these modifications from otherwise normal RNA bases, which we were able to discover from the visualization.
Conclusions: Sequoia's interactive features complement existing computational approaches in nanopore-based RNA workflows. The insights gleaned through visual analysis should help users in developing rationales, hypotheses, and insights into the dynamic nature of RNA. Sequoia is available at https://github.com/dnonatar/Sequoia .
Keywords: Bioinformatics; Epitranscriptome; Nanopore signal analysis; Next-generation-sequencing; RNA modifications; Single-Molecule Sequencing; Visual infrastructure.