Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications

Nanopore direct RNA sequencing is a technology that allows sequencing for epitranscriptomic modifications with the possibility of a quantitative assessment. In the present work, pseudouridine (Ψ) was sequenced with the nanopore before and after the pH 7 bisulfite reaction that yields stable ribose adducts at C1′ of Ψ. The adducted sites produced greater base call errors in the form of deletion signatures compared to Ψ. Sequencing studies on E. coli rRNA and tmRNA before and after the pH 7 bisulfite reaction demonstrated that using chemically-assisted nanopore sequencing has distinct advantages for minimization of false positives and false negatives in the data. The rRNA from E. coli has 19 known U/C sequence variations that give similar base call signatures as Ψ, and therefore, are false positives when inspecting base call data; however, these sites are refractory to reacting with bisulfite as is easily observed in nanopore data. The E. coli tmRNA has a low occupancy Ψ in a pyrimidine-rich sequence context that is called a U representing a false negative; partial occupancy by Ψ is revealed after the bisulfite reaction. In a final study, 5-methylcytidine (m 5 C) in RNA can readily be observed after the pH 5 bisulfite reaction in which the parent C deaminates to U and the modified site does not react. This locates m 5 C when using bisulfite-assisted nanopore direct RNA sequencing, which is otherwise challenging to observe. The advantages and challenges of the overall approach are discussed. Nanopore direct RNA sequencing assisted by pseudouridine- and m 5 C-specific bisulfite treatment is a technology that allows sequencing for epitranscriptomic modifications with the possibility of quantitative assessment.