Abstract
Transcriptome profiling of RNA modifications is essential for uncovering and characterizing novel post-transcriptional regulatory mechanisms. However, most of the known RNA modifications remain poorly explored due to technological limitations. Nanopore direct RNA sequencing (DRS) provides an advantageous solution for transcriptome-wide RNA modification profiling, enabling simultaneous identification of any modification type in native RNA, with full-length coverage and single-molecule resolution. We developed RMPore (https://rmpore.renlab.cn/), a comprehensive database of single-molecule RNA modifications detected from 958 DRS samples across 34 species. We constructed a practical analytical pipeline integrating 20 detection tools and categorized all detected sites into three confidence levels (high, medium, and low) based on the prediction thresholds and reproducibility of tools, datasets, and other technologies, identifying a total of 65 025 784 modification sites spanning 25 modification types. To further investigate the characteristics of these modification sites and elucidate the regulatory relationships among different modification types, we performed single-molecule advanced analyses of correlated sites and haplotype-biased sites. Meanwhile, we also incorporated extensive molecular event annotations of modification sites in RMPore, including splicing events, RNA-binding protein interactions, RNA-RNA interactions, and circular RNAs. We expect that RMPore will advance single-molecule epitranscriptomics research, bridging critical gaps in the field of RNA modification research.