Performance and practicality of 16S nanopore sequencing for routine bacterial identification in clinical samples

The 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 times 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. We 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 (CE-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. Comparative analysis revealed high concordance between NGS platforms across the clinical cohort, achieving a weighted species overlap of 93.5 ± 7.6% and a Cohen’s 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 h less total processing time, and proving more cost-effective for small-batch, on-demand diagnostics than the Illumina workflow. This 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.

IMPORTANCE

This study validates a 16S nanopore sequencing (ONT) pipeline against the gold-standard Illumina workflow for accurate bacterial identification in low-diversity clinical samples, essential for effective diagnostics. The pipeline showed high consistency with Illumina across 101 clinical samples. Crucially, the ONT workflow proved significantly more cost-effective and demonstrated superior efficiency, requiring approximately 50 h less total time, making it feasible for routine clinical implementation. The impact for routine diagnostics was made possible by the combination of ONT’s game-changing technology with the Molzym CE IVD-marked products using ultra-pure PCR reagents and tests. We conclude that 16S nanopore sequencing is a feasible, time- and cost-effective method suitable for direct implementation in routine clinical diagnostic workflows.