RNA modifications detection by comparative Nanopore direct RNA sequencing

Adrien Leger, Paulo P. Amaral, Luca Pandolfini, Charlotte Capitanchik, Federica Capraro, Valentina Miano, Valentina Migliori, Patrick Toolan-Kerr, Theodora Sideri, Anton J. Enright, Konstantinos Tzelepis, Folkert J. Werven, Nicholas M. Luscombe, Isaia Barbieri, Jernej Ule, Tomas Fitzgerald, Ewan Birney (), Tommaso Leonardi () and Tony Kouzarides ()
Additional contact information
Adrien Leger: Wellcome Genome Campus, Hinxton
Paulo P. Amaral: University of Cambridge, Tennis Court Road
Luca Pandolfini: University of Cambridge, Tennis Court Road
Charlotte Capitanchik: The Francis Crick Institute
Federica Capraro: The Francis Crick Institute
Valentina Miano: Division of Cellular and Molecular Pathology, University of Cambridge
Valentina Migliori: University of Cambridge, Tennis Court Road
Patrick Toolan-Kerr: The Francis Crick Institute
Theodora Sideri: The Francis Crick Institute
Anton J. Enright: University of Cambridge, Tennis Court Road
Konstantinos Tzelepis: University of Cambridge, Tennis Court Road
Folkert J. Werven: The Francis Crick Institute
Nicholas M. Luscombe: The Francis Crick Institute
Isaia Barbieri: University of Cambridge, Tennis Court Road
Jernej Ule: The Francis Crick Institute
Tomas Fitzgerald: Wellcome Genome Campus, Hinxton
Ewan Birney: Wellcome Genome Campus, Hinxton
Tommaso Leonardi: University of Cambridge, Tennis Court Road
Tony Kouzarides: University of Cambridge, Tennis Court Road

Nature Communications, 2021, vol. 12, issue 1, 1-17

Abstract: Abstract RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.

Date: 2021
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DOI: 10.1038/s41467-021-27393-3

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