Using Nanocompore to Identify RNA Modifications from Direct RNA Nanopore Sequencing Data

AbstractRNA modifications can alter the behavior of RNA molecules depending on where they are located on the strands. Traditionally, RNA modifications have been detected and characterized by biophysical assays, mass spectrometry, or specific next‐generation sequencing techniques, but are limited to specific modifications or are low throughput. Nanopore is a platform capable of sequencing RNA strands directly, which permits transcriptome‐wide detection of RNA modifications. RNA modifications alter the nanopore raw signal relative to the canonical form of the nucleotide, and several software tools detect these signal alterations. One such tool is Nanocompore, which compares the ionic current features between two different experimental conditions (i.e., with and without RNA modifications) to detect RNA modifications. Nanocompore is not limited to a single type of RNA modification, has a high specificity for detecting RNA modifications, and does not require model training. To use Nanocompore, the following steps are needed: (i) the data must be basecalled and aligned to the reference transcriptome, then the raw ionic current signals are aligned to the sequences and transformed into a Nanocompore‐compatible format; (ii) finally, the statistical testing is conducted on the transformed data and produces a table of p‐value predictions for the positions of the RNA modifications. These steps can be executed with several different methods, and thus we have also included two alternative protocols for running Nanocompore. Once the positions of RNA modifications are determined by Nanocompore, users can investigate their function in various metabolic pathways. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: RNA modification detection by NanocomporeAlternate Protocol 1: RNA modification detection by Nanocompore with f5cAlternate Protocol 2: RNA modification detection by Nanocompore using Nextflow