A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing

Summary Antimicrobial resistance (AMR) poses a significant threat to public health. Rapid and accurate antimicrobial sensitivity testing is essential to guide effective treatment. Here, we present “simplified 5PSeq” (s5PSeq), a streamlined protocol for profiling 5’ monophosphorylated (5’P) mRNA degradation intermediates that reflect ribosome dynamics in vivo. By capturing antibiotic-induced, context-specific ribosome stalling events, s5PSeq provides a molecular proxy for bacterial growth inhibition—offering a molecular phenotypic readout without the need for culturing. s5PSeq reduces library preparation time to under four hours and incorporates a novel rRNA blocking strategy. We demonstrated its clinical utility by identifying erythromycin-resistant and sensitive Clostridioides difficile clinical isolates. Combining s5PSeq with real-time nanopore sequencing enables fast AMR diagnosis with as few as 3000 reads. In addition to simplifying the study of 5’P co-translational mRNA decay, our work suggests that utilizing information-rich phenotypic molecular readouts can significantly improve AMR diagnostics. Highlights s5PSeq is a streamlined protocol for profiling 5’P mRNA degradation intermediates. Context-specific ribosome stalls can be used to assess phenotypic antimicrobial sensitivity at the molecular level. Blocking rRNA sequencing at the ligation step streamlines library preparation and lowers costs and hands-on time. Integration with nanopore sequencing allows same-day antimicrobial sensitivity testing in species with 5’-3’ exonuclease. Competing Interest Statement VP, SH & LN are co-founders and RH COO of 3N Bio AB which has a patent application regarding part of the work described in this manuscript. XY is the founder of RocRock Biotechnology. The rest of authors declare no competing interest. Footnotes ↵8 Lead contact