De novo basecalling of RNA modifications at single molecule and nucleotide resolution

Cruciani, Sonia; Delgado-Tejedor, Anna; Pryszcz, Leszek Piotr, 1985-; Medina, Rebeca; Llovera Nadal, Laia; Novoa, Eva Maria

doi:http://dx.doi.org/10.1186/s13059-025-03498-6

De novo basecalling of RNA modifications at single molecule and nucleotide resolution

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Cruciani S, Delgado-Tejedor A, Pryszcz LP, Medina R, Llovera L, Novoa EM. De novo basecalling of RNA modifications at single molecule and nucleotide resolution. Genome Biol. 2025 Feb 25;26(1):38. DOI: 10.1186/s13059-025-03498-6

Abstract

RNA modifications influence RNA function and fate, but detecting them in individual molecules remains challenging for most modifications. Here we present a novel methodology to generate training sets and build modification-aware basecalling models. Using this approach, we develop the m6ABasecaller, a basecalling model that predicts m6A modifications from raw nanopore signals. We validate its accuracy in vitro and in vivo, revealing stable m6A modification stoichiometry across isoforms, m6A co-occurrence within RNA molecules, and m6A-dependent effects on poly(A) tails. Finally, we demonstrate that our method generalizes to other RNA and DNA modifications, paving the path towards future efforts detecting other modifications.