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.
Highlights
The occurrence of m6A in eukaryotic polyadenylated RNA is among the most investigated phenomena in recent RNA research
While adenosines are largely inert to nucleophiles like bisulfite, we found reports on adenosine deamination by nitrous acid [43], which led to the characterization of N6-methyl-N6-nitrosoadenosine (NOm6A) [44] (Figure 1A), with unknown base-pairing properties
In order to distinguish m6A from unmethylated adenosines in sequencing data, the major step was the conversion of adenosines into inosines to change the base-pairing properties in reverse transcription
Summary
The occurrence of m6A in eukaryotic polyadenylated RNA is among the most investigated phenomena in recent RNA research. The community is in agreement, that the use of antibodies alone, or in refining combinations with other techniques [15,16,17], still does not provide quantitative data at single nucleotide resolution [28]. Numerous tools from chemical biology have been applied to the task, including catalytic DNA [29], chemically altered dNTPs [30], engineered SAM analogues [31] or engineered polymerases [32] for the generation of a reverse transcription signature, or derivatives of SAM or metabolic precursor methionine [33] for click chemistry-based enrichment. Current developments focus on the use of nanopore technology [34],
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