Mateos, P. AceraSethi, A. J.Ravindran, A.Srivastava, A.Woodward, K.Mahmud, S.Kanchi, M.Guarnacci, M.Xu, J.Yuen, Z. W. S.Zhou, Y.Sneddon, A.Hamilton, W.Gao, J.Starrs, L. M.Hayashi, R.Wickramasinghe, V.Zarnack, K.Preiss, T.Burgio, G.Dehorter, N.Shirokikh, N. E.Eyras, E.2025-05-302025-05-302041-1723PubMed:38724548WOS:001217774000002ORCID:/0009-0009-5304-924X/work/164350440ORCID:/0000-0001-6273-784X/work/164351063ORCID:/0000-0001-8249-358X/work/164351137ORCID:/0000-0002-8567-9168/work/164351368ORCID:/0000-0003-0793-6218/work/164351541ORCID:/0000-0002-0117-6898/work/164354321ORCID:/0000-0002-5848-9019/work/168142701http://www.scopus.com/inward/record.url?scp=85192568706&partnerID=8YFLogxKhttps://hdl.handle.net/1885/733754716The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI’s capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.We are grateful to the personnel from the Biomolecular Resource Facility at JCSMR (ANU), and particularly to Tiffany Cripps, Lachlan Morrison, Carolina Correa Ospina and Stephanie Palmer, for their assistance with DNA validation sequencing. We are also grateful to the personnel of the Ecogenomics and Bioinformatics Lab, a joint initiative of the Research School of Biology (ANU) and Commonwealth Scientific and Industrial Research Organisation, and particularly to Niccy Aitken and Ashley Jones for their continued support and feedback regarding ONT sequencing. We acknowledge funding support by the Australian Research Council (ARC) Discovery Project grants DP220101352 (to E.E. and T.P.), DP210102385 (to T.P., R.H. and E.E.), and DP180100111 (to T.P. and N.S.); by the National Health and Medical Research Council (NHMRC) Senior Research Fellowship APP1135928 (to T.P.), Investigator Grant GNT1175388 (to N.S.), and Ideas Grant 2018833 (to E.E.). This research was also indirectly supported by the Australian Government\u2019s National Collaborative Research Infrastructure Strategy (NCRIS) through access to computational resources provided by the National Computational Infrastructure (NCI) through the National Computational Merit Allocation Scheme (NCMAS), the ANU Merit Allocation Scheme (ANUMAS), and Phenomics Australia. The funding bodies had no role in study design, data collection, or data analysis.17enPublisher Copyright: © The Author(s) 2024.5-methylcytosineEnrichmentDna2024-05-0910.1038/s41467-024-47953-785192568706