Nationwide multicentre study of Nanopore long-read sequencing for 16S rRNA-species identification.
| Title: | Nationwide multicentre study of Nanopore long-read sequencing for 16S rRNA-species identification. |
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| Authors: | Brunet S; Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. sofia.brunet@gu.se.; Dept of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Region Västra Götaland, Sweden. sofia.brunet@gu.se.; Grankvist A; Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.; Dept of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Region Västra Götaland, Sweden.; Jaen-Luchoro D; Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.; Dept of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Region Västra Götaland, Sweden.; Bergdahl M; Department of Clinical Microbiology, Centrallasarettet, Växjö, Region Kronoberg County, Sweden.; Tison JL; Department of Clinical Microbiology, Centralsjukhuset i Karlstad, Karlstad, Region Värmland County, Sweden.; Wester A; Department of Clinical Microbiology, Centralsjukhuset i Karlstad, Karlstad, Region Värmland County, Sweden.; Elfving K; Department of Clinical Microbiology, Falu Lasarett, Region Dalarna County, Falun, Sweden.; Brandenburg J; Department of Clinical Microbiology, Falu Lasarett, Region Dalarna County, Falun, Sweden.; Gullsby K; Department of Clinical Microbiology, Gävle Sjukhus, Gävle, Region Gävleborg County, Sweden.; Lindsten C; Department of Clinical Microbiology, Hallands Sjukhus Halmstad, Halmstad, Region Halland County, Sweden.; Arvidsson LO; Department of Clinical Microbiology, Hallands Sjukhus Halmstad, Halmstad, Region Halland County, Sweden.; Larsson H; Department of Clinical Microbiology, Länssjukhuset Kalmar, Kalmar, Region Kalmar County, Sweden.; Eilers H; Department of Clinical Microbiology, Norrlands Universitetssjukhus, Umeå, Region Västerbotten County, Sweden.; Strand AS; Department of Clinical Microbiology, Skåne University Hospital, Lund, Region Skåne County, Sweden.; Lannefors M; Center for Molecular Diagnostics, Skåne University Hospital, Lund, Region Skåne County, Sweden.; Keskitalo J; Department of Clinical Microbiology, Sunderby Sjukhus, Luleå, Region Norrbotten County, Sweden.; Rylander F; Department of Clinical Microbiology, Sundsvalls Sjukhus, Sundsvall, Region Västernorrland County, Sweden.; Welander J; Department of Clinical Microbiology, and Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden.; Jungestrom MB; Department of Clinical Microbiology, and Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden.; Geörg M; Department of Laboratory Medicine, Västmanland Hospital, Västerås, Region Västmanland County, Sweden.; Kaden R; Department of Medical Sciences, Clinical Microbiology, Uppsala University, 751 85, Uppsala, Sweden.; Clinical Genomics Uppsala, Science for Life Laboratory, Uppsala University, 751 85, Uppsala, Sweden.; Karlsson I; Clinical Genomics Uppsala, Science for Life Laboratory, Uppsala University, 751 85, Uppsala, Sweden.; Department of Immunology, Genetics and Pathology, Uppsala University, 751 85, Uppsala, Sweden.; Linde AM; Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.; Mernelius S; Laboratory Medicine, Jönköping and Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Region Jönköping County, Sweden.; Berglind L; Laboratory Medicine, Jönköping and Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Region Jönköping County, Sweden.; Feuk L; National Genomics Infrastructure, Uppsala University, Uppsala, Sweden.; Kerje S; National Genomics Infrastructure, Uppsala University, Uppsala, Sweden.; Karlsson L; Swedish Defence Research Agency - FOI, Umeå, Sweden.; Sjödin A; Swedish Defence Research Agency - FOI, Umeå, Sweden.; Guerra-Blomqvist L; Department of Clinical Microbiology, Karolinska University Hospital and Karolinska Institute, Region Stockholm County, Stockholm, Sweden.; Wallin F; Department of Laboratory Medicine, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.; Fagerström A; Clinical Genomics, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.; Vondracek M; Department of Clinical Microbiology, Karolinska University Hospital and Karolinska Institute, Region Stockholm County, Stockholm, Sweden.; Mölling P; Department of Laboratory Medicine, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.; Hallbäck ET; Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.; Dept of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Region Västra Götaland, Sweden. |
| Source: | European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology [Eur J Clin Microbiol Infect Dis] 2025 Aug; Vol. 44 (8), pp. 1907-1916. Date of Electronic Publication: 2025 May 10. |
| Publication Type: | Journal Article; Multicenter Study |
| Language: | English |
| Journal Info: | Publisher: Springer Country of Publication: Germany NLM ID: 8804297 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1435-4373 (Electronic) Linking ISSN: 09349723 NLM ISO Abbreviation: Eur J Clin Microbiol Infect Dis Subsets: MEDLINE |
| Imprint Name(s): | Publication: Berlin : Springer; Original Publication: [Wiesbaden, Federal Republic of Germany] : Vieweg, [c1988- |
| MeSH Terms: | RNA, Ribosomal, 16S*/genetics ; Nanopore Sequencing*/methods ; Bacteria*/genetics ; Bacteria*/classification ; Bacteria*/isolation & purification ; Bacterial Infections*/diagnosis ; Bacterial Infections*/microbiology; DNA, Bacterial/genetics ; Sequence Analysis, DNA/methods ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Sweden ; Nanopores ; Computational Biology |
| Abstract: | Purpose: Recent improvements in Nanopore sequencing chemistry has made it a promising platform for long-read 16S rRNA sequencing. This study evaluated its clinical utility in a nationwide collaboration coordinated by Genomic Medicine Sweden.; Methods: Thirteen mock samples comprised of various bacterial strains and an External Quality Assessment (EQA) panel from QCMD (Quality Control for Molecular Diagnostics) were analysed by 20 microbiological laboratories across Sweden, using the recent v14 chemistry. Most laboratories generated full-length 16S rRNA sequencing libraries using an optimized protocol for the 16S Barcoding Kit 24, while two laboratories employed in-house PCR coupled with the Ligation Sequencing Kit. The commercial 16S bioinformatic pipeline from 1928 Diagnostics (1928-16S) was evaluated and compared with the open-sourced gms_16S pipeline that is based on the EMU classification tool (GMS-16S).; Results: Seventeen out of 20 laboratories successfully sequenced and analysed the samples. Laboratories that used sodium acetate-containing elution buffers faced compatibility issues during library construction, resulting in reduced read count. High bacterial load samples were generally well-characterized, whereas hard-to-lyse bacteria such as Gram-positive strains were detected at lower abundance. The GMS-16S tool provided improved species-level identification compared to the 1928-16S pipeline, particularly for closely related taxa within the Streptococcus and Staphylococcus genera.; Conclusion: Nanopore sequencing demonstrated promising potential for bacterial identification in a clinical setting. The results prompt further optimization of the protocol to improve detection of a broader range of species. This multicentre study highlights the feasibility of implementing Nanopore sequencing into clinical microbiological laboratories, for improved national precision diagnostics.; (© 2025. The Author(s).) |
| Competing Interests: | Declarations. Competing interest: The authors declare no competing interests. |
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| Contributed Indexing: | Keywords: 16S rRNA; Bacterial identification; EMU; Genomic Medicine Sweden; Nanopore sequencing |
| Substance Nomenclature: | 0 (RNA, Ribosomal, 16S); 0 (DNA, Bacterial) |
| Entry Date(s): | Date Created: 20250510 Date Completed: 20250805 Latest Revision: 20250807 |
| Update Code: | 20260130 |
| PubMed Central ID: | PMC12321653 |
| DOI: | 10.1007/s10096-025-05158-w |
| PMID: | 40348924 |
| Database: | MEDLINE |
Journal Article; Multicenter Study