Direct RNA sequencing enables m(6)A 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 N-6-methyladenosine (m(6)A) sites within DRACH motifs. Our software MINES (m(6)A Identification using Nanopore Sequencing) assigned m(6)A 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 m(6)A writer, METTL3, and eraser, ALKBH5, respectively. MINES (https://github.com/YeoLab/MINES.git) enables m(6)A annotation at single coordinate-level resolution from direct RNA nanopore sequencing.