Abstract
Nanopore direct RNA sequencing (DRS) coupled with Dorado modification-aware basecalling enables mapping of epitranscriptomic modifications including N (6) -methyladenosine (m (6) A) at the level of individual RNAs. However, a lack of systematic benchmarking continues to raise questions regarding the sensitivity, specificity, and reproducibility of this method. To address this and to establish a best-practice workflow, we evaluated multiple Dorado versions using in vitro transcribed RNA and an m (6) A methyltransferase inhibitor as specificity controls. We established that stringent filtering is necessary to reduce false-positive calls and found strong concordance at high-stoichiometry sites when compared to an orthogonal m (6) A mapping method (GLORI). Further, by applying DRS to primary human fibroblasts and HD10.6 neurons, we uncovered cell type-specific differences in m (6) A stoichiometry, indicating a finely tuned epitranscriptomic regulation. Our study thus presents the first systematic comparison of Dorado and GLORI from the same input RNA and expands characterization of the m (6) A epitranscriptome to fibroblasts and neurons.