A possible alternative to the detection of epitranscriptomic modifications by SMRT sequencing is the direct detection using the Nanopore sequencing technologies. This technique exploits nanometer-sized protein channels embedded into a membrane or solid materials, and coupled to sensors, able to detect the amplitude and duration of the variations of the ionic current passing through the pore. As the RNA passes through the nanopore, the blockage leads to a disruption in current stream, which is different for the different bases, included modified ones, and therefore can be used to identify possible modifications. By producing single-molecule reads, without previous RNA amplification and conversion to cDNA, these techniques can lead to the production of quantitative transcriptome-wide maps.
In particular, the Nanopore technology proved to be effective in detecting the presence of two nucleotide analogs in RNA: N-methyladenosine (m6A) and 5-methylcytosine (5-mC). Using Hidden Markov Models (HMM) or recurrent neural networks (RNN) trained with known sequences, it was possible to demonstrate that the modified nucleotides produce a characteristic disruption in the ionic current when passing through the pore, and that these data can be used to identify the nucleotide.