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bioRxiv · 10.1101/2025.11.12.687992

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

Liu, H.·Huch, S.·Hull, R.·Romero Garcia, F.·Nersisyan, L.·Yin, X.·Chen, W.-H.·Chen, J.·Pelechano, V.
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Abstract

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 (5P) 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 5P co-translational mRNA decay, our work suggests that utilizing information-rich phenotypic molecular readouts can significantly improve AMR diagnostics. HighlightsO_LIs5PSeq is a streamlined protocol for profiling 5P mRNA degradation intermediates. C_LIO_LIContext-specific ribosome stalls can be used to assess phenotypic antimicrobial sensitivity at the molecular level. C_LIO_LIBlocking rRNA sequencing at the ligation step streamlines library preparation and lowers costs and hands-on time. C_LIO_LIIntegration with nanopore sequencing allows same-day antimicrobial sensitivity testing in species with 5-3 exonuclease. C_LI

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Liu, H., Huch, S., Hull, R., Romero Garcia, F., Nersisyan, L., Yin, X., Chen, W.-H., Chen, J., Pelechano, V.. 2025-11-12. A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing. https://doi.org/10.1101/2025.11.12.687992

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