Towards Swedish national precision diagnostics | ESCMID Global 2026

Abstract: Background: Advances in Oxford Nanopore Technologies’ sequencing chemistry have strengthened the platform’s potential for long-read 16S rRNA sequencing for clinical bacterial identification. Three Swedish university hospitals (Sahlgrenska, Örebro and Karolinska) jointly evaluated the beta version of the recently released Microbial Amplicon Barcoding Sequencing kit for 16S and ITS (SQK-MAB114.24). The assessment included a diverse set of bacterial species, 15 clinical specimen types, and mock samples containing defined bacterial strains.

Methods: Full-length 16S rRNA libraries were prepared utilizing the beta-SQK-MAB114.24 kit, with an increased number of PCR cycles to adopt the protocol for low level clinical samples. Libraries were sequenced on R10 flow cells with MinION or GridION instruments v14 using chemistry. Data were analyzed using the open-source Taxonomic Reconstruction and Analysis of NGS Amplicons (TRANA) pipeline, which incorporates the EMU taxonomic classifier.

Results: The workflow detected bacterial loads as low as 15 CFU/mL for both gram-positive (Staphylococcus aureus) as well as gram-negative (Klebsiella pneumoniae) strains in pleura mock samples. A total of 84 monomicrobial and 14 polymicrobial clinical samples and bacterial strains, representing diverse taxonomic groups were analyzed, alongside 17 negative samples. Findings were overall concordant with routine in-house methods (Sanger or Ion Torrent sequencing) at the participating laboratories. TRANA provided species-level resolution within closely related genera such as Streptococcus and Staphylococcus.

Conclusion: Nanopore full-length 16S rRNA sequencing demonstrated promising potential for fast and accurate bacterial identification in a clinical setting. The results support continued validation of the protocol, including broader assessment of primer specificity, sensitivity and workflow robustness in clinical laboratories.