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RNA modifications detection by comparative Nanopore direct RNA sequencing

View ORCID ProfileAdrien Leger, View ORCID ProfilePaulo P. Amaral, View ORCID ProfileLuca Pandolfini, View ORCID ProfileCharlotte Capitanchik, View ORCID ProfileFederica Capraro, View ORCID ProfileIsaia Barbieri, Valentina Migliori, View ORCID ProfileNicholas M. Luscombe, View ORCID ProfileAnton J Enright, Konstantinos Tzelepis, View ORCID ProfileJernej Ule, View ORCID ProfileTomas Fitzgerald, View ORCID ProfileEwan Birney, View ORCID ProfileTommaso Leonardi, View ORCID ProfileTony Kouzarides
doi: https://doi.org/10.1101/843136
Adrien Leger
European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
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  • ORCID record for Adrien Leger
Paulo P. Amaral
The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UKStorm Therapeutics Limited, Moneta Building B280, Babraham Research Campus, Cambridge CB22 3AT, UK
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  • ORCID record for Paulo P. Amaral
Luca Pandolfini
The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
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  • ORCID record for Luca Pandolfini
Charlotte Capitanchik
The Francis Crick Institute, London NW1 1AT, UK
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  • ORCID record for Charlotte Capitanchik
Federica Capraro
The Francis Crick Institute, London NW1 1AT, UKDepartment of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, Queen Square, London, WC1N 3BG, UK
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  • ORCID record for Federica Capraro
Isaia Barbieri
The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UKDepartment of Pathology, Division of cellular and molecular pathology, lab block level 3, box 231, Addenbrooke’s hospital, CB2 0QQ, Cambridge, UK
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Valentina Migliori
The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
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Nicholas M. Luscombe
The Francis Crick Institute, London NW1 1AT, UKDepartment of Genetics, Environment and Evolution, UCL Genetics Institute, London, UKOkinawa Institute of Science & Technology Graduate University, Okinawa, Japan
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Anton J Enright
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK
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  • ORCID record for Anton J Enright
Konstantinos Tzelepis
The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
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Jernej Ule
The Francis Crick Institute, London NW1 1AT, UKDepartment of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, Queen Square, London, WC1N 3BG, UK
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Tomas Fitzgerald
European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
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  • ORCID record for Tomas Fitzgerald
Ewan Birney
European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
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  • ORCID record for Ewan Birney
  • For correspondence: t.kouzarides@gurdon.cam.ac.uk tommaso.leonardi@iit.it birney@ebi.ac.uk
Tommaso Leonardi
The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UKCenter for Genomic Science, Italian Institute of Technology (IIT), Milan, Italy
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  • For correspondence: t.kouzarides@gurdon.cam.ac.uk tommaso.leonardi@iit.it birney@ebi.ac.uk
Tony Kouzarides
The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
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  • ORCID record for Tony Kouzarides
  • For correspondence: t.kouzarides@gurdon.cam.ac.uk tommaso.leonardi@iit.it birney@ebi.ac.uk
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Abstract

RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. To date, over 150 naturally occurring PTMs have been identified, however the overwhelming majority of their functions remain elusive. In recent years, a small number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing (DRS) technology has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework to evaluate the presence of modifications in DRS data. To do so, we compare an RNA sample of interest against a non-modified control sample. Our strategy does not require a training set and allows the use of replicates to model biological variability. Here, we demonstrate the ability of Nanocompore to detect RNA modifications at single-molecule resolution in human polyA+ RNAs, as well as in targeted non-coding RNAs. Our results correlate well with orthogonal methods, confirm previous observations on the distribution of N6-methyladenosine sites and provide novel insights into the distribution of RNA modifications in the coding and non-coding transcriptomes. The latest version of Nanocompore can be obtained at https://github.com/tleonardi/nanocompore.

Footnotes

  • ↵* Co-first authors

  • https://github.com/tleonardi/nanocompore

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted November 15, 2019.
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RNA modifications detection by comparative Nanopore direct RNA sequencing
Adrien Leger, Paulo P. Amaral, Luca Pandolfini, Charlotte Capitanchik, Federica Capraro, Isaia Barbieri, Valentina Migliori, Nicholas M. Luscombe, Anton J Enright, Konstantinos Tzelepis, Jernej Ule, Tomas Fitzgerald, Ewan Birney, Tommaso Leonardi, Tony Kouzarides
bioRxiv 843136; doi: https://doi.org/10.1101/843136
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RNA modifications detection by comparative Nanopore direct RNA sequencing
Adrien Leger, Paulo P. Amaral, Luca Pandolfini, Charlotte Capitanchik, Federica Capraro, Isaia Barbieri, Valentina Migliori, Nicholas M. Luscombe, Anton J Enright, Konstantinos Tzelepis, Jernej Ule, Tomas Fitzgerald, Ewan Birney, Tommaso Leonardi, Tony Kouzarides
bioRxiv 843136; doi: https://doi.org/10.1101/843136

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