Abstract
The chemical modification of ribonucleotides plays an integral role in the biology of diverse viruses and their eukaryotic host cells. Mapping the precise identity, location, and abundance of modified ribonucleotides remains a key goal of many studies aimed at characterizing the function and importance of a given modification. While mapping of specific RNA modifications through short-read sequencing approaches has powered a wealth of new discoveries in the past decade, this approach is limited by inherent biases and an absence of linkage information. Moreover, in viral contexts, the challenge is increased due to the compact nature of viral genomes giving rise to many overlapping transcript isoforms that cannot be adequately resolved using short-read sequencing approaches. The recent emergence of nanopore sequencing, specifically the ability to directly sequence native RNAs from virus-infected host cells, provides not just a new methodology for mapping modified ribonucleotides but also a new conceptual framework for what can be derived from the resulting sequencing data. In this minireview, we provide a detailed overview of how nanopore direct RNA sequencing works, the computational approaches applied to identify modified ribonucleotides, and the core concepts underlying both. We further highlight recent studies that have applied this approach to interrogating viral biology and finish by discussing key experimental considerations and how we predict that these methodologies will continue to evolve.