RNA modifications detection by comparative Nanopore direct RNA sequencing.

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Amaral, Paulo P 
Capitanchik, Charlotte 

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.

Base Sequence, Computational Biology, Gene Expression Profiling, Genetic Techniques, High-Throughput Nucleotide Sequencing, Humans, Nanopore Sequencing, Nanopores, RNA, RNA Processing, Post-Transcriptional, Sequence Analysis, RNA, Software, Transcriptome
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Nat Commun
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Springer Science and Business Media LLC
Wellcome Trust (092096/Z/10/Z)
Cancer Research UK (A27321)
Cancer Research UK (17001)
Cancer Research UK (C7/A18061)
Cancer Research UK (C6946/A24843)
Wellcome Trust (203144/Z/16/Z)
Biotechnology and Biological Sciences Research Council (BB/K017438/1)
Wellcome Trust (203144/A/16/Z)
The Kouzarides laboratory is supported by Cancer Research UK (grant reference RG72100) and core support from the Wellcome Trust (core grant reference WT203144) and Cancer Research UK (grant reference C6946/A24843). PPA was supported by a Borysiewicz Biomedical Sciences postdoctoral fellowship (University of Cambridge) and AL by a COFUND Marie Skłodowska-Curie Actions postdoctoral fellowship (EMBL). FW and TS are supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001203), the UK Medical Research Council (FC001203), and the Wellcome Trust (FC001203). IB and V Miano are supported by Cancer Research UK (grant reference RG86786) and by the Joseph Mitchell Fund.