{"paper_id":"01b8cadd-a8a8-49e3-a663-4134d72b0b2f","body_text":"Abstract\nDirect RNA sequencing with Oxford Nanopore Technologies (ONT) captures nucleotide-specific current signals that reflect both sequence and chemical modifications, offering the potential to detect RNA modifications directly from native RNA molecules. To interpret such signals, ONT provides modification-aware basecalling models that estimate the probability of selected modifications at each nucleotide. In May 2025, ONT released updated modification-calling models (Dorado v5.2.0) for pseudouridine (Ψ), inosine, m6A and m5C, alongside new models for 2′O-ribose-methylations, necessitating independent validation. Here, we benchmark Dorado v5.2.0 against v5.1.0 using ex cellulo tRNAs from Schizosaccharomyces pombe, leveraging their well-defined modification landscape. We generated modification probability profiles at single-nucleotide resolution and quantified model performance using curated sets of annotated and validated modification sites. Our results reveal that, despite notable improvements in Ψ detection, most modification callers remain challenged by the dense and heterogeneous modification environments of tRNAs. This work provides the first comprehensive evaluation of Dorado v5.2.0 on native tRNAs and establishes a methodological framework for benchmarking future ONT modification models in complex RNA modification contexts.\nCompeting Interest Statement\nThe authors have declared no competing interest.","source_license":"CC-BY-4.0","license_restricted":false}