Campodónico Victoria L, Ruelle Jean, Fitzgerald Anna, Bergman Yehudit, Osborne Brenda, Bourdas Dimitrios, Lu Jennifer, Carroll Karen C, Simner Patricia J
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
SmartGene Services, EPFL Innovation Park, Lausanne, Switzerland.
J Clin Microbiol. 2025 May 14;63(5):e0167024. doi: 10.1128/jcm.01670-24. Epub 2025 Apr 22.
Sanger sequencing of the first 500 bp of the 16S rRNA gene is frequently used to identify bacterial pathogens that have ambiguous biochemical profiles or proteomic mass spectra. When diversity does not occur within that region, genus-level and/or species-level identification may not be possible, and a longer sequence or alternative target may be required to distinguish between genera/species. In this study, we evaluated a clinically relevant end-to-end solution for long-read (1,500 nt) 16S rRNA next-generation sequencing by Oxford Nanopore Technologies (ONT) compared to a ~500 nt Sanger sequencing approach for the identification of 153 bacterial clinical isolates. Sequencing data were analyzed using the IDNS software from SmartGene and its proprietary 16S Centroid reference database (Centroid database) SmartGene software and the Centroid database. The agreement of the two platforms on species- and genus-level identification was determined, and discrepancies were resolved by whole-genome sequencing. ONT had a higher taxonomic resolution at the genus level ( < 0.01). When genus-level identification was achieved by both methods, concordance to the best matching genus was 100%. When species-level identification was achieved by both methods, concordance to the best matching species was 91%. The costs per test were ~$25.30 (when multiplexing 24 samples/run) and $74 for ONT and Sanger sequencing, respectively. The hands-on time spent performing sequencing was similar for both methods, but the turnaround time of ONT was significantly shorter than that of Sanger sequencing.IMPORTANCEThis study adds to existing literature by describing a validated end-to-end solution of 16S rRNA gene Oxford Nanopore sequencing for bacterial isolate identification, including sequencing run time evaluation, automated analysis (SmartGene 16S Identification App) and interpretation of results, that can be incorporated into clinical and public health laboratories with a simple and cost-effective workflow.
16S rRNA基因前约500 bp的桑格测序常用于鉴定生化特征或蛋白质组质谱不明确的细菌病原体。当该区域内不存在多样性时，可能无法进行属级和/或种级鉴定，可能需要更长的序列或替代靶点来区分不同的属/种。在本研究中，我们评估了一种临床相关的端到端解决方案，用于通过牛津纳米孔技术（ONT）进行长读长（约1500 nt）16S rRNA下一代测序，并与约500 nt的桑格测序方法进行比较，以鉴定153株细菌临床分离株。使用来自SmartGene的IDNS软件及其专有的16S质心参考数据库（质心数据库）SmartGene软件和质心数据库对测序数据进行分析。确定了两个平台在种级和属级鉴定上的一致性，并通过全基因组测序解决差异。ONT在属级具有更高的分类分辨率（<0.01）。当两种方法都实现属级鉴定时，与最佳匹配属的一致性为100%。当两种方法都实现种级鉴定时，与最佳匹配种的一致性为91%。每次检测的成本分别为ONT约25.30美元（当每次运行多重检测24个样本时）和桑格测序74美元。两种方法进行测序所花费的实际操作时间相似，但ONT的周转时间明显短于桑格测序。重要性本研究通过描述一种经过验证的用于细菌分离株鉴定的16S rRNA基因牛津纳米孔测序的端到端解决方案，包括测序运行时间评估、自动化分析（SmartGene 16S鉴定应用程序）和结果解读，为现有文献增添了内容，该解决方案可以通过简单且经济高效的工作流程纳入临床和公共卫生实验室。
