Probing the epitranscriptome and RNA damage with nanopore direct RNA sequencing

Abstract

Nanopore direct RNA sequencing (DRS) is revolutionizing our ability to analyze the epitranscriptome to evaluate nucleoside modifications in both cellular and synthetic RNA. The process involves minimal handling of fragile RNA strands, one round of reverse transcription to provide a DNA:RNA duplex, and library preparation to directly read nucleotides with their modifications as they pass through a protein nanopore embedded in a membrane. Simultaneous sequencing of hundreds of strands on a chip provides unprecedented access to whole transcriptome information. A key advantage is the long-read length that permits, for example, operon-specific epitranscriptomics of ribosomal RNA modifications as a function of cellular stress. By analyzing the entire transcriptome, the interplay of different modifications on the same RNA, or the correlation of changes in different RNAs in the same cell type, can be monitored. This review presents several recent examples of the types of experiments that are suitable for nanopore DRS as well as some of the current challenges and future expectations.