Quantification of Pseudourdine in human ribosomal RNA by direct RNA sequencing
Pseudouridine is the most abundant post-transcriptional modification in human ribosomal RNA (rRNA), with over 110 annotated sites including a subset with variable stoichiometry across biological contexts. Existing quantification methods are either low-throughput or constrained to predefined target panels, limiting systematic profiling. Here, we benchmark nanopore direct RNA sequencing using the Dorado v5.1 basecalling model against mass spectrometry–validated pseudouridine sites in rRNA from human liver tissue, induced pluripotent stem cells, and HeLa cells, using HydraPsiSeq as an orthogonal comparator. Nanopore sequencing detected 95 of 117 mass spectrometry–validated sites and accurately quantified stoichiometry at 85% of sites with high reproducibility across batches and biological contexts. Sites in low guanine-cytosine sequence environments were identified as the primary source of quantification failure. Compared to HydraPsiSeq, nanopore sequencing showed markedly lower batch variability and enabled site-agnostic detection. These results establish nanopore direct RNA sequencing as a scalable and reproducible tool for rRNA pseudouridine profiling, with defined thresholds guiding its application in epitranscriptomic studies.