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bioRxiv · 10.64898/2026.06.08.730855

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.
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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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S de Preval, B., Faucher-Giguere, L., Duval, M., Marchand, V., Lakshmi Narasimha, P., Thakor, N., Motorin, Y., Abou Elela, S., Scott, M. S.. 2026-06-08. Nanopore Direct RNA Sequencing Enables Reproducible, Site-Resolved Pseudouridine Quantification in Human Ribosomal RNA. https://doi.org/10.64898/2026.06.08.730855

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