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

Nanopore direct RNA sequencing (DRS) coupled with Dorado modification-aware basecalling enables mapping of epitranscriptomic modifications including N6-methyladenosine (m6A) at the level of individual RNAs. However, the sensitivity, specificity, and reproducibility of this method remain unclear and have only recently begun to be addressed through systematic benchmarking studies. Here, we aimed to establish a best-practice workflow for DRS-based epitranscriptomic analyses. Specifically, we evaluated multiple Dorado versions and models using RNA isolated from primary cells and unmodified in vitro transcribed RNAs. We further utilized an m6A methyltransferase inhibitor as a specificity control. We established that stringent filtering is necessary to reduce the contribution of background and found that Dorado predictions captured an increasing proportion of GLORI sites detected at high m6A/A proportions. Further, by applying DRS to a human primary fibroblast and a HD10.6 neuron dataset, we detected differences in the predicted m6A/A proportions at conserved sites between these datasets. Our study thus presents the first systematic comparison of Dorado and GLORI from the same input RNA and expands characterization of the m6A epitranscriptome to fibroblasts and neurons.