Validation of a long-read 16S rRNA-gene sequencing approach for analysis of clinical samples and bacterial identification in a routine clinical laboratory.

Background: Currently, microbiological diagnosis relies heavily on classical culture-based techniques, which have notable limitations. These methods often struggle with detecting non-culturable or slow-growing microorganisms and can yield negative results in patients who have already received antimicrobial treatment before sample collection. 16S rRNA gene sequencing offers an alternative to culture-based methods, enabling bacterial identification without the need for cultivation. This study presents the validation and clinical implementation of a 16S rRNA-gene Nanopore-based sequencing workflow in a routine hospital. Methods: Estimated limit of detection and linearity were investigated using spiking experiments with a dilution series of ATCC strains and commercial quality control material. Accuracy and including taxonomic resolution was evaluated for 37 strains obtained from external quality control programs and 30 clinical from a clinical collection. Utility was investigated by comparing 16S rRNA gene Nanopore-based sequencing with culture results and other clinical findings for 54 prospectively collected clinical samples. Results: An estimated limit of detection of 260 and 747 CFU/ml was found for respectively Discussion: This study demonstrates that the implementation of 16S rRNA gene sequencing is feasible in a routine clinical laboratory setting and provides additional utility for the identification of bacterial isolates and direct detection on clinical samples, increasing recovery of non-culturable isolates.