Single-Molecule Direct RNA Sequencing Reveals the Shaping of Epitranscriptome Across Multiple Species

RNA modifications play a crucial role in gene regulation, yet understanding their transcriptome-wide landscape and biogenesis mechanisms remains challenging. Traditional next-generation sequencing (NGS) methods detect RNA modifications based on the aggregation of short-reads, overlooking the nature of RNA as multiple transcripts from one gene. Third-generation sequencing (TGS) platforms provide direct RNA sequencing at the resolution of individual RNA molecules, offering the promise of detecting RNA modifications and RNA processing events simultaneously. In this study, we introduce SingleMod, a deep learning model tailored for precise m6A modification mapping on individual RNA molecules using nanopore direct RNA sequencing (DRS). We systematically dissect the transcriptome-wide m6A landscape at single-molecule and single-base resolution, refining our understanding of the genomics of m6A and revealing an additive mode through which m6A shapes the epitranscriptome. Through comparative analyses across diverse species, we quantitatively elucidate three distinct m6A distribution patterns that suggest diverse biogenesis mechanisms. This study pioneers single-molecule epitranscriptome exploration across multiple species, deepening our understanding of m6A, including its genomics, biogenesis, mechanisms, and biological implications. Absolute quantification of m6A in mouse embryonic stem cells, Arabidopsis, Rice, and Chamydomonas using GLORI. Single-molecule level m6A profiling of HEK293T, Chamydomonas and Rice using nanopore direct RNA sequencing.