Chemical Probe-Based Nanopore Sequencing to Selectively Assess the RNA Modifications

Abstract

Nanopore direct RNA sequencing (dRNA-Seq) reads reveal RNA modifications through consistent error profiles specific to a modified nucleobase. However, a null data set is required to identify actual RNA modification-associated errors for distinguishing it from confounding highly intrinsic sequencing errors. Here, we reveal that inosine creates a signature mismatch error in dRNA-Seq reads and obviates the need for a null data set by harnessing the selective reactivity of acrylonitrile for validating the presence of actual inosine modifications. Selective reactivity of acrylonitrile toward inosine altered multiple dRNA-Seq parameters like 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.

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing

“…To overcome this bottleneck, chemical decorations would assist in making m6A more distinguishable from unmodified A when traversing the nanopore. Similar methods have been developed to detect inosine using the nanopore sequencing 43 .…”

Nanopore sequencing for the 17 modification types in 36 locations inE. coliribosomal RNA enables monitoring of stress-dependent changes

“…Nevertheless, chemical modifications to RNA can be identified by their unique signatures compared to the canonical forms in the base call, ionic current, and/or dwell time data as they pass the helicase active site and then through the constriction zone of the pore. 10-17…”

“…Modifications to RNA resulting in base calling differences have enabled sequencing for pseudouridine (Ψ), N 6 -methyladenine (m 6 A), inosine (I), the 2′- O -methylation of A, C, G, and U, inosine, and N7-methylguanosine (m 7 G), as examples. 10-17 Some RNA modifications, such as 5-methylcytidine (m 5 C) do not strongly impact the base calling and are not easily found by this method. 13 Alternatively, the ionic current level can differ for RNA modifications, such as Ψ, as it passes through the nanopore and can be analyzed to determine the frequency of a modification.…”

Detection of nucleotide modifications in bacteria and bacteriophages: Strengths and limitations of current technologies and software

“…As discussed above, BS‐seq requires a pretreatment of the samples to detect methylations and some other minor modifications may lead to only small changes in signal on third generation sequencing platforms. In a recent preprint study (Ramasamy et al, 2020), the chemical probe acrylonitrile was used to selectively cyanoethylate inosine and pseudouridine (Ψ) RNA modifications in an epitranscriptome obtained with Nanopore. These RNA modifications are otherwise difficult and laborious to detect with single nucleotide resolution, but are important to the functional role of RNAs, for example, ensuring mRNA stability, transcriptional repression, and translational efficiency.…”