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

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.

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

“…Recently, the global deamination of all RNA bases by nitrous acid treatment was used for m 6 A mapping and quantification. The protocol, termed NOSeq [64], exploits the resistance of m 6 A to chemical deamination under conditions where all other RNA nucleotides are efficiently converted. The protocol allows for targeted sequencing for the confirmation of m 6 A's presence and the relative quantification of the modified residue.…”

Long Non-Coding RNA Epigenetics

“…Nitrous acid deaminates adenosines to inosine, while the m6A residue is resistant to such modifications. The application of NGS after modification to detect m6A sites in MALAT1 lncRNA [ 130 ].…”

“…The detection of the m 6 A nucleotide in RNA, using an indirect approach, is difficult because few chemical reagents modify the methyl group. However, NOseq, a method for the detection of m 6 A in RNA after chemical deamination by nitrous acid, has recently been introduced [130]. Nitrous acid deaminates adenosines to inosine, while the m6A residue is resistant to such modifications.…”

General Principles for the Detection of Modified Nucleotides in RNA by Specific Reagents

“…[67,98] As illustrated in Figure 5, the deamination of adenosines by nitric acid in NOseq is based on the generation of an NO + electrophile from nitrite under acidic conditions, which converts exocyclic amino groups into leaving groups. [99] Because this reagent leads to the deamination of all exocyclic amino groups, there is comprehensive chemical A-to-I and C-to-U editing in RNA, which makes data treatment after Illumina sequencing more complicated than for bisulfite seq. Modifications, e.g., m 6 A stall the deamination reaction on the level of the respective nitroso compound which inhibits A-to-I editing.…”

“…While we are certain that several brands of vintage organic chemistry still await their application to deep sequencing, one must be aware that some reagents might simply not be selective enough for application to full-fledged epitranscriptomes, despite being able to discriminate short modified versus unmodified RNAs. [17,22] Rather, it might be helpful to the community to determine the size of a "limited transcriptome," [99] such as, e.g., the collective of tRNAs and rRNAs of ≈10 4 nucleotides, that can be meaningfully investigated with a given combination of reagent and reaction conditions. For example, at false positive rate of 1 in 1000, we expect 10 false positives in transcriptome of that size, which is a manageable number for validation by orthogonal methods.…”