Charting the epitranscriptomic landscape across RNA biotypes using native RNA nanopore sequencing

RNA modifications are conserved chemical features found in all domains of life and across diverse RNA biotypes, shaping gene expression profiles and enabling rapid responses to environmental changes. Their broad chemical diversity and dynamic nature pose significant challenges for studying them comprehensively. These limitations can now be addressed through direct RNA nanopore sequencing (DRS), which allows simultaneous identification of diverse RNA modification types at single-molecule and single-nucleotide resolution. Here, we review recent efforts pioneering the use of DRS to better understand the epitranscriptomic landscape. We highlight how DRS can be applied to investigate different RNA biotypes, emphasizing the use of specialized library preparation protocols and downstream bioinformatic workflows to detect both natural and synthetic RNA modifications. Finally, we provide a perspective on the future role of DRS in epitranscriptomic research, highlighting remaining challenges and emerging opportunities from improved sequencing yields and accuracy enabled by the latest DRS chemistry.