Full-Length 16S rRNA Amplicon Sequencing for the Simple and Simultaneous Detection of Multiple Probiotic Species in Commercial Products

To ensure efficacy and trust, probiotic strains in commercial products should be accurately identified. However, studies have consistently reported discrepancies between labeling and actual microbial composition. Therefore, we developed a full-length 16S rRNA amplicon sequencing workflow for species-level identification of probiotics. To optimize this workflow, four mock communities were analyzed using multiple pipelines and parameters; the workflow accuracy was evaluated. Optimal performance was achieved using Q10 filtering, the Emu pipeline with its default database, and a 0.1% relative abundance threshold. Subsequently, the workflow was applied to eight probiotic products, approximately 50% of which revealed labeling discrepancies with some labeled strains undetected. Furthermore, these results were compared and confirmed using PCR analysis. Despite some discrepancies, both approaches demonstrated overall consistency. Overall, the optimized sequencing workflow facilitates comprehensive species-level identification of probiotics within a single assay, providing a novel approach for quality control and labeling verification.