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 over 13,000 previously unannotated DRACH sites in endogenous HEK293T transcripts and identified over 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, ALKHBH5, respectively.

MINES (https://github.com/YeoLab/MINES.git) enables long-read direct RNA-seq to m6A annotation at single coordinate-level resolution.