Detection of ribonucleotides embedded in DNA by Nanopore sequencing

Ribonucleotides represent the most common non-canonical nucleotides found in eukaryotic genomes which can lead to negative consequences. High-throughput sequencing technologies available to map rNMPs in DNA only allow to indirectly deduce their genomic location. The sequencing tools provided by Oxford Nanopore Technologies (ONT) may overcome such limitation, helping to unmask the real sites of rNMPs incorporation in genomic DNA directly from raw sequencing signals. We assessed the feasibility of detecting ribonucleotides embedded in synthetic DNA substrates by ONT generating linear double stranded DNA molecules containing rNMPs at known positions and we developed appropriate data analysis pipelines. We report for the first time that the Nanopore machinery can identify all four ribonucleotides incorporated in DNA by capturing rNMPs-related alterations in nucleotide alignments, current features, and dwell times. These findings constitute the proof of concept that ONT may successfully be used to directly detect and map rNMPs embedded in genomic DNA.