Accurate detection of m6A RNA modifications in native RNA sequences [Curlcake constructs]

The field of epitranscriptomics has experienced a major revolution in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here we show that using the platform offered by Oxford Nanopore Technologies, we can detect N6-methyladenosine (m6A) RNA modifications with high accuracy. Our results open new avenues to investigate the universe of RNA modifications with single nucleotide, single molecule resolution.   Overall design: For synthesis purposes, a total of 4 sequences were designed by splitting the 10kb sequence into smaller sequences of slightly different size (2329bp, 2543bp, 2678bp and 2795bp, which we named 'Curlcake 1', 'Curlcake 2', 'Curlcake 3' and 'Curlcake 4', respectively). These constructs were in-vitro transcribed either in the presence of ATP or N6-Methyladenosine-5'-Triphosphate (m6ATP). Sequencing was performed with two replicates of two libraries containing non-modified RNAs in one flowcell and m6ATP-modified RNAs in the second flowcell, giving a total of 4 samples. The FASTA file for the sequences is provided as a supplementary file.