6533b827fe1ef96bd1286699
RESEARCH PRODUCT
NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination
Jean-yves RoignantJean-yves RoignantMaksim V. SednevAndreas HildebrandtTina LenceFlorian PichotMark HelmAurellia GalliotVirginie MarchandThomas KemmerJulian KönigYuri MotorinClaudia HöbartnerStephan Wernersubject
AdenosineSequence analysisAcademicSubjects/SCI00010Bisulfite sequencingDeaminationAdenosine/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 SpectrometrySequence alignmentComputational biologyBiology010402 general chemistry[SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry Molecular Biology/Molecular biology01 natural sciencesTranscriptome03 medical and health sciencesNarese/13Tandem Mass Spectrometry[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry Molecular Biology/Genomics [q-bio.GN]GeneticsRNA Ribosomal 18SAnimalsHumansRNA MessengerComputingMilieux_MISCELLANEOUS030304 developmental biology0303 health sciencesSequence Analysis RNARNAHigh-Throughput Nucleotide Sequencing[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry Molecular Biology/Molecular biologyAmpliconRibosomal RNA0104 chemical sciencesDrosophila melanogasterHEK293 CellsDeaminationMethods OnlineRNA[SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry Molecular Biology/Genomics [q-bio.GN]RNA Long NoncodingSequence AlignmentAlgorithmsChromatography LiquidHeLa Cellsdescription
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.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2020-12-11 |