This project is a continuation of the NAISS project 2025/22-159, “Nanopore-based diagnosis of phenotypic antimicrobial resistance,” which resulted in the development and publication of a streamlined, nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial susceptibility testing (Liu et al., Cell Reports Methods, 2026). This method enables genome-wide profiling of mRNA decay as a functional readout of bacterial responses to antibiotic exposure, capturing condition-specific RNA degradation signatures that directly reflect phenotypic susceptibility. Building on this foundation, the current project aims to extend and validate the s5PSeq approach in clinically relevant settings. Specifically, we will apply the method to bacterial populations derived from patient blood culture samples in collaboration with the Blomqvist laboratory at Karolinska Institutet. The primary objective is to evaluate the robustness and generalizability of s5PSeq-derived phenotypic signatures across multiple antibiotic classes and heterogeneous clinical samples, thereby advancing the method toward clinical applicability. The experimental workflow involves short antibiotic exposure (10 minutes) followed by RNA extraction and s5PSeq library preparation. Particular emphasis will be placed on optimizing sample preparation from complex clinical matrices, including mitigation of inhibitory effects associated with blood-derived components. In parallel, established computational pipelines will be adapted and scaled to support systematic analysis of clinical datasets, enabling identification of antibiotic-specific RNA decay signatures and classification of susceptibility phenotypes. The study is expected to include approximately 100 samples, each sequenced to a depth of ~5 million reads using Illumina platforms. Data collection is anticipated to be completed by mid-2026 and will support a manuscript demonstrating the translational potential of s5PSeq as a rapid, phenotypic antimicrobial susceptibility testing framework. Although samples originate from patient blood cultures, bacterial enrichment is performed prior to sequencing, resulting in minimal human-derived sequence content. All data will be processed within secure NAISS infrastructure, and any residual human reads will be removed during preprocessing. The project complies with approved ethical protocols (dnr 2024-03500-01), ensuring that all samples are de-identified and handled in accordance with institutional and regulatory requirements.