Epitranscriptomic modifications are emerging as important factors in fine-tuning gene expression and regulation. To date, more than 170 distinct RNA chemical modifications have been characterised, thereby increasing the complexity of eukaryotic transcriptomes. Although next-generation sequencing platforms have contributed to deciphering several modifications, such as A-to-I RNA editing—the most abundant base conversion in humans—charting the entire epitranscriptome remains challenging.

Oxford Nanopore Sequencing Technology (ONT) offers a unique opportunity to sequence native RNA molecules (dRNA-seq), preserving epitranscriptomic modifications and enabling single-molecule, high-resolution profiling of RNA modifications. Analysing dRNA-seq data requires modification-aware base-calling algorithms that implement complex deep learning models, while raw current signals demand several preprocessing steps for downstream analyses.

This section provides a foundation for experimental planning, analytical strategies, and computational approaches to applying Nanopore direct RNA sequencing for profiling RNA modifications.