Nucleic Acids Res.: co-auth.: J-Y.Roignant

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Nucleic Acids Res. 2020 Dec 11;gkaa1173. doi: 10.1093/nar/gkaa1173. Online ahead of print.

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination

Stephan Werner 1, Aurellia Galliot 1, Florian Pichot 1, Thomas Kemmer 2, Virginie Marchand 3, Maksim V Sednev 4, Tina Lence 5, Jean-Yves Roignant 1 5 6, Julian König 5, Claudia Höbartner 4, Yuri Motorin 7, Andreas Hildebrandt 2, Mark Helm 1Affiliations expand

Abstract

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.