Calibrated analysis framework for nanopore direct RNA sequencing uncovers cell-specific m6A proportions at conserved sites

Calibrated nanopore DRS analysis reveals cell-specific m6A proportions at conserved sites, with Dorado predictions capturing increasing GLORI site proportions at high m6A/A levels.

• Why it matters: Understanding the accuracy and reproducibility of nanopore DRS for epitranscriptomic modifications is crucial for reliable m6A mapping, yet systematic benchmarking has been limited.
• What they did: The study evaluated multiple Dorado versions and models using primary cell and in vitro transcribed RNAs, incorporating an m6A methyltransferase inhibitor as a specificity control, and applied stringent filtering to improve prediction accuracy.
• The result: Differences in predicted m6A/A proportions at conserved sites were observed between human fibroblast and neuron datasets, expanding the characterization of the m6A epitranscriptome across cell types.