show Abstracthide AbstractPseudouridine (?) is an abundant mRNA modification in the mammalian transcriptome, but its function has remained elusive due to the difficulty of transcriptome-wide mapping. We develop nanopore native RNA sequencing for quantitative ? analysis that utilizes native content training, machine learning model prediction, and single read coordination. We find interferon inducible ? modifications in the interferon stimulated gene transcripts, consistent with a role of ? in the efficacy of mRNA vaccines. Overall design: Illumina seq of rRNA mixture from human, yeast, C.elegans, Drosophila and stool microbiome