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 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.