Resolving systematic bias in nanopore-based RNA modification detection
Sneddon, A.; Prodic, S.; Eyras, E.
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Nanopore direct RNA sequencing has accelerated the growth of the epitranscriptomics field, yet nanopore-based RNA modification landscapes are generally unreliable due to the heuristic and unvalidated quantification algorithm underpinning the standard, widely used method, modkit. We introduce a general benchmarking framework that uncovers systematic and often extreme bias in modkits default implementation, which misleads biological conclusions, and we identify optimised settings that rescue modkit performance across multiple modification types and biological contexts, establishing an accurate and reliable standard for RNA modification quantification. A Nextflow workflow is available at https://github.com/comprna/modkitopt.
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