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bacterial genome sequencing, libraries from different strains are usually multiplexed in a single run. Normalized libraries are most often pooled in equal volumes, as recommended by next-generation sequencing platform manufacturers. This equal-volume strategy is well suited for multiplexing isolates from the same species. However, for runs involving multiple microbial species an equimolar library pooling is more adapted because of the variation in bacterial genome size. To demonstrate its utility in clinical microbiology, we compared both equal-volume and equimolar strategies using a menu comprising 13 bacterial species involved in healthcare-associated infections. We show that equimolar pooling limits the retesting risk due to insufficient coverage depth, particularly when inter-species genome size difference is more than 2-fold. The use of this alternative strategy for multiplexing pathogenic bacteria should lead to more cost effective whole-genome sequencing applications in clinical microbiology.xref:PubMed:31227316ENALibrary, species.falseEquimolar NGS library pooling for multiplexing bacterial species2023-05-192019-04-02PRJNA49082031227316