Direct RNA sequencing enables m⁶A detection in endogenous transcript isoforms at base-specific resolution

Direct RNA sequencing holds great promise for the de novo identification of RNA modifications at single-coordinate resolution; however, interpretation of raw sequencing output to discover modified bases remains a challenge. Using Oxford Nanopore's direct RNA sequencing technology, we developed a random forest classifier trained using experimentally detected N⁶-methyladenosine (m⁶A) sites within DRACH motifs. Our software MINES (m⁶A Identification using Nanopore Sequencing) assigned m⁶A methylation status to more than 13,000 previously unannotated DRACH sites in endogenous HEK293T transcripts and identified more than 40,000 sites with isoform-level resolution in a human mammary epithelial cell line. These sites displayed sensitivity to the m⁶A writer, METTL3, and eraser, ALKBH5, respectively. MINES (https://github.com/YeoLab/MINES.git) enables m⁶A annotation at single coordinate-level resolution from direct RNA nanopore sequencing.