Where's Wally? Finding RNA modifications with ONT direct-RNA Sequencing

RNA molecules undergo a vast array of post-transcriptional modifications (PTMs). Transcriptome-wide PTM mapping methods such as RNA-immunoprecipitation and chemoselective alteration have revolutionised the field, however they are laborious and there are concerns about their lack of reproducibility. To evaluate the ability of ONT direct RNA sequencing to detect PTMs, we developed a protocol to concatenate <em>in vitro</em> synthesised RNA blocks containing a single modified nucleotide (m⁶A or m⁶2A) surrounded by all possible sequence contexts. We analysed our direct RNA-Seq data by integrating signal intensity, dwell time and local sequence context. Our descriptive statistical analysis revealed that the presence of m⁶A and m⁶2A induces a distinctive change in the signal for the majority of the k-mers. We are now working on extending our PTM catalogue and are actively developing a deep learning based PTM detection package