Performance and Practicality of 16S Nanopore Sequencing for Routine Bacterial Identification in Clinical Samples

BackgroundThe accurate and timely identification of bacterial pathogens in low-diversity samples is critical for clinical diagnostics, yet traditional culture-based methods often fail due to prior antibiotic exposure or fastidious growth requirements. While 16S rRNA gene sequencing provides a culture-independent alternative, traditional Sanger sequencing cannot resolve polymicrobial infections, and short-read sequencing platforms are often limited by long turnaround time and high associated costs. Oxford Nanopore Technologies (ONT) offers a promising solution through rapid turnaround times and lower costs; however, its clinical adoption is hindered by a lack of standardized, validated protocols and large-scale comparative data. MethodsWe developed a rapid, cost-effective 16S rRNA diagnostic workflow using ONT and benchmarked its performance against an Illumina Next-Generation Sequencing (NGS) workflow. The pipeline utilizes an in vitro diagnostic (IVD) certified amplification protocol targeting the V3-V4 region with DNA-free reagents to minimize contamination in low-biomass specimens. We first verified the detection limits using dilution series of pure and mixed bacterial cultures. Subsequently, the workflow was prospectively applied to 101 clinical samples, with results evaluated retrospectively against Illumina data and subjected to rigorous clinical review to determine infection plausibility. ResultsComparative analysis revealed high concordance between NGS platforms across the clinical cohort, achieving a weighted species overlap of 93.5 {+/-} 7.6% and a Cohens kappa of 0.81 {+/-} 0.04 upon clinical review. While Illumina demonstrated slightly higher sensitivity at the lowest microbial concentrations in dilution series experiments, ONT generated sequences with comparable average accuracy (99.9 {+/-} 0.39% for ONT, 99.8 {+/-} 0.42% for Illumina). Crucially, the ONT workflow was significantly more efficient, requiring approximately 50 hours less total processing time and proving more cost-effective for small-batch, on-demand diagnostics than the Illumina workflow. ConclusionsThis study offers strong supporting evidence for the integration of 16S nanopore sequencing into routine infectious disease diagnostics. Our findings demonstrate that 16S nanopore sequencing is a feasible, time-efficient, and high-resolution alternative to established NGS methods, particularly suited for rapid, decentralized clinical implementation for diagnostic sequencing of low-diversity samples.