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

## TL;DR

This paper introduces a new method to detect two RNA modifications, m6A and pseudouridine, in single RNA molecules using third-generation sequencing.

## Contribution

The paper introduces Ψ-co-mAFiA, an updated method that detects both m6A and pseudouridine modifications simultaneously at single-molecule resolution.

## Key findings

- Ψ-co-mAFiA detects m6A in all 18 DRACH contexts and pseudouridine sites in single RNA molecules.
- Modification level predictions align well with orthogonal methods and reflect changes in writer protein activity.
- The method enables the study of cross-modification interactions on individual 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 An archived version of the software is available on Zenodo at https://doi.org/10.5281/zenodo.16797676.