PurposeRecent 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.MethodsThirteen 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).ResultsSeventeen 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.ConclusionNanopore 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.