Nanopore Direct RNA Sequencing Enables Reproducible, Site-Resolved Pseudouridine Quantification in Human Ribosomal RNA
S de Preval, B.; Faucher-Giguere, L.; Duval, M.; Marchand, V.; Lakshmi Narasimha, P.; Thakor, N.; Motorin, Y.; Abou Elela, S.; Scott, M. S.
Show abstract
Pseudouridine is the most abundant post-transcriptional modification in human ribosomal RNA, with over 110 annotated sites and variable stoichiometry across biological contexts. Existing quantification methods are low-throughput or constrained to predefined panels. We benchmarked nanopore direct RNA sequencing using the Dorado v5.1 model against mass spectrometry-validated sites in human liver tissue, induced pluripotent stem cells, and HeLa cells. Nanopore sequencing detected 95 of 117 validated sites and accurately quantified stoichiometry at 85% of sites with high reproducibility. Low GC-content environments were the primary source of failure. These results establish nanopore sequencing as a scalable tool for epitranscriptomic pseudouridine profiling.
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