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

Details

Serval ID
serval:BIB_3CBA60432C31
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.
Journal
Nucleic acids research
Author(s)
Werner S., Galliot A., Pichot F., Kemmer T., Marchand V., Sednev M.V., Lence T., Roignant J.Y., König J., Höbartner C., Motorin Y., Hildebrandt A., Helm M.
ISSN
1362-4962 (Electronic)
ISSN-L
0305-1048
Publication state
Published
Issued date
26/02/2021
Peer-reviewed
Oui
Volume
49
Number
4
Pages
e23
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't ; Validation Study
Publication Status: ppublish
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.
Keywords
Adenosine/analogs & derivatives, Adenosine/analysis, Algorithms, Animals, Chromatography, Liquid, Deamination, Drosophila melanogaster/genetics, HEK293 Cells, HeLa Cells, High-Throughput Nucleotide Sequencing/methods, Humans, RNA/chemistry, RNA, Long Noncoding/chemistry, RNA, Messenger/chemistry, RNA, Ribosomal, 18S/chemistry, Sequence Alignment, Sequence Analysis, RNA/methods, Tandem Mass Spectrometry
Pubmed
Open Access
Yes
Create date
12/12/2020 14:57
Last modification date
17/03/2021 7:26
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