Calibrated analysis framework for nanopore direct RNA sequencing uncovers cell-specific m ⁶ A stoichiometry at conserved sites

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 human primary 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.