ABSTRACTThe Oxford Nanopore MinION sequencing platform offers direct analysis of DNA reads as they are generated, which combined with its low cost, low power and extremely compact size, makes the device attractive for in-field or clinical deployment, e.g. rapid diagnostics. We employed the MinION platform for shotgun metagenomic sequencing and analysis of mixed gut-associated microbial communities; firstly, we used a 20 species human microbiota mock community to show that Nanopore metagenomic data can be classified reliably and rapidly. Secondly, we profiled bacterial DNA isolated from faeces from preterm infants at increased risk of sepsis and necrotising enterocolitis to analyse their gut microbiota. Using longitudinal samples, and comparing Illumina to MinION, we captured the diversity of the immature gut microbiota and observed how its complexity changes over time in response to interventions, i.e. probiotic, antibiotics and episodes of suspected sepsis. Finally, we performed a 'real-time' run from sample to analysis using a faecal sample of a critically ill infant. Real-time analysis was facilitated by our new NanoOK RT software package. We determined that we can reliably identify potentially pathogenic taxa (i.e. Klebsiella pneumoniae) along with corresponding AMR gene profiles in as little as one hour, post sequencing start. Furthermore, data obtained revealed insights into how antibiotic treatment decisions may be rapidly modified in response to specific AMR profiles, which was validated using pathogen isolation, whole genome sequencing and antibiotic susceptibility testing. Our results demonstrate that MinION sequencers offer the ability to progress from clinical samples to a potential tailored patient antimicrobial treatment in just a few hours.