NOseq: amplicon sequencing evaluation method for RNA m(6)A sites after chemical deamination

Methods for the detection of m(6)A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m(6)A 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, m(6)A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m(6)A immunoprecipitation. NOseq faithfully detected known m(6)A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m(6)A candidate sites, drawn from miCLIP data with an m(6)A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m(6)A sites in defined sequences.