Ψ-co-mAFiA: concurrent detection of pseudouridine and m6A in single RNA molecules.

Abstract

The development of third-generation sequencing technologies enables the detection of RNA modifications at single-molecule resolution. Specifically for direct RNA sequencing on the ONT platform, we have previously developed an m6A detection algorithm called mAFiA. Here, we present the updated method, now covering all 18 DRACH m6A contexts as well as the identification of pseudouridine sites (Ψ). Our modification level predictions compare favorably with orthogonal methods and respond to knockdown or knock out of writer proteins. The simultaneous detection of multiple modifications on a single RNA molecule opens up the possibility to study cross-modification interactions. Ψ-co-mAFiA is available at https://github.com/dieterich-lab/psi-co-mAFiA and licensed under GPLv3.0. An archived version of the software is available on Zenodo at https://doi.org/10.5281/zenodo.16797676.