ON-Time enables rapid microbiome sequencing and analysis for precision medicine.

Clinical application of microbiome-guided therapies in the intensive care unit (ICU) requires a method to rapidly analyze patient microbiomes to guide urgent treatment decisions. Conventional microbiome sequencing and analysis methods require long turnaround times, methodological complexity, and high costs that are barriers to clinical application. Here, we describe a method for rapid (<5 h turnaround from sample to results) and accurate metagenomic sequencing and taxonomic analysis of microbiomes in individual patient fecal samples, called ON-Time. ON-Time uses a simplified and rapid wet-lab workflow coupled with point-and-click data analysis. Accuracy and precision of ON-Time data were validated using defined mock microbial communities and head-to-head comparison with conventional shotgun metagenomics of ICU patient samples. Key limitations include stochastic identification of functional genes such as antimicrobial resistance and virulence factors. Taken together, ON-Time offers a rapid, accurate, and cost-effective method to analyze individual patient samples for clinically actionable microbiome features to guide personalized therapeutics.