Rapid and reliable species-level identification from clinical samples using 16 S rRNA gene nanopore sequencing analysis

The detection and identification of bacterial species in clinical samples are crucial for patient management and antibiotic treatment. When culture-based identification methods fail, 16 S rRNA gene next-generation sequencing (NGS) serves as a valuable alternative. However, its clinical utility is often limited by prolonged time to results (TtR) and limited species-level resolution. This study aimed to develop and validate a faster, more discriminative 16 S rRNA gene NGS workflow. Our current 16 S rRNA gene NGS protocol uses micelle-based PCR (micPCR) targeting the V4 region, followed by Illumina sequencing. This method ensures accurate quantification of 16 S rRNA gene copies in (low biomass) clinical samples by reducing PCR artefacts and correcting for background DNA contamination. To shorten the TtR and improve species-level determination, the micPCR protocol was adapted to amplify full-length 16s rRNA genes, followed by nanopore sequencing using the Flongle Flow Cell with automated data analysis using the Genome Detective platform. Testing with a synthetic microbial community and six clinical samples showed that the 16 S rRNA gene micPCR/nanopore sequencing protocol maintains good accuracy and sensitivity, reducing TtR to 24 h and enhancing species-level resolution. This optimized workflow improves clinical diagnostics, making it a valuable tool for guiding patient treatment decisions.