Acrylonitrile, Inosine and nanopore sequencing. Chemical Probe-Based Nanopore Sequencing to Selectively Assess the RNA Modification

Nanopore direct RNA sequencing (dRNA-Seq) reads reveal RNA modifications through consistent error profiles specific to a modified nucleobase. However, a null dataset is required to identify actual RNA modification-associated errors for distinguishing it from confounding high intrinsic sequencing errors. Here, we reveal that inosine created signature mismatch error in dRNA-Seq reads and obviated the need for a null dataset by harnessing the selective reactivity of acrylonitrile for validating the presence of actual inosine modifications. Selective reactivity of acrylonitrile towards inosine altered multiple dRNA-Seq parameters like insertion frequency, signal intensity and trace value. We also deduced the stoichiometry of inosine modification through deviation in signal intensity and trace value using this chemical biology approach. Furthermore, we devised Nano ICE-Seq, a protocol to overcome the low coverage issue associated with direct RNA sequencing. Taken together, our chemical probe-based approach may facilitate the knockout-free detection of disease-associated RNA modifications in clinical scenarios