Abstract
Bloodstream infections and sepsis necessitate rapid, sensitive, and clinically relevant diagnostics to minimize treatment delays and improve clinical outcomes. Next-generation sequencing enables culture-independent pathogen detection, antimicrobial resistance profiling, and genome-informed epidemiology. This narrative review integrates clinical evidence with practical workflows across three complementary approaches. We describe the use of plasma microbial cell-free DNA for broad organism detection and burden monitoring, as well as metagenomic next-generation sequencing of blood or plasma for unbiased pathogen discovery, including culture-negative and polymicrobial infections. Same-day Oxford Nanopore Technologies sequencing of positive blood culture broth is also discussed as a way to accelerate species identification, targeted resistance reporting, and infection-prevention decisions. We outline the sample-to-result steps, typical turnaround time (TAT), and stewardship-aligned decision points. Analytical reliability depends on effective reduction in human DNA background, stringent control of background and reagent-derived nucleic acids in low-biomass samples, and documented and validated bioinformatics workflows that are supported by curated taxonomic and resistance databases. Quantitative reports should adhere to validated thresholds and should be interpreted in the context of internal controls and clinical pretest probability. Ongoing challenges include variable correlation between genotype and phenotype for specific pathogen and antibiotic pairs, interpretation of low-level signals, and inconsistent regulatory and reimbursement environments. Advances in portable sequencing, faster laboratory and analytical workflows, and scaled liquid biopsy strategies may further reduce the TAT and expand access. Integrating these tools within One Health frameworks and global genomic surveillance programs could support early resistance detection and coordinated public health action, which could help to advance sepsis care toward more precise treatment and real-time infection control insights.