A streamlined, nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing
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 (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 4 h 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 3,000 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.
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BibTeX
@article{liu2026streamlined,
title = {{A streamlined, nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing}},
author = {Honglian Liu and Susanne Huch and Ryan Hull and Fabricio Romero Garcia and Lilit Nersisyan and Xiushan Yin and Wei-Hua Chen and Juan Du and Vicent Pelechano},
journal = {Cell Reports Methods},
year = {2026},
month = oct,
doi = {10.1016/j.crmeth.2026.101327},
url = {https://hal.science/hal-05780642v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - A streamlined, nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing
AU - Honglian Liu
AU - Susanne Huch
AU - Ryan Hull
AU - Fabricio Romero Garcia
AU - Lilit Nersisyan
AU - Xiushan Yin
AU - Wei-Hua Chen
AU - Juan Du
AU - Vicent Pelechano
PY - 2026
DA - 2026/10/06
JO - Cell Reports Methods
DO - 10.1016/j.crmeth.2026.101327
UR - https://hal.science/hal-05780642v1
AB - 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 4 h 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 3,000 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.
SN - 2667-2375
SN - 2667-2375
ER -