Xiao Fei ¹ Zengzhi Yuan ² Sandra Marina Wellner ¹ Yibing Ma ¹ John Elmerdahl Olsen ^1*

• ¹ University of Copenhagen, Copenhagen, Denmark
• ² Tianjin Normal University, Tianjin, China

Advancements in next-generation sequencing (NGS) have significantly accelerated the development of innovative methodologies in microbiological research. In this study, we present a novel method to quantify the net survival of gene-deletion mutants within the intracellular environment. Based on standardized Illumina short-read sequencing of genomic DNA, the method eliminates the need for specific selective markers on each deletion mutant.For validation, the method was shown to accurately quantify mutants in spiked pools of mixed mutants, showing no statistically significant differences compared to the expected values based on CFU determination (P >0.05). Further, the method was used to quantify mutants of S.Gallinarum in macrophages. Six mutants and one control strain were mixed in a pool and allowed to infect HD11 cells for two hours. The results align with prior research results, providing evidence of the feasibility of mixed mutant infections in functional gene identification. Notably, the simplicity and standardization of the method, rooted in standard whole-genome sequencing protocols, make it easily implementable across various laboratories.The study presents a novel method for quantifying the intracellular net-survival of mutants in mixed pools of gene-deletion mutants without the need for distinct selective marker on each mutant. This innovative approach, validated with S. Gallinarum infection of macrophages, offers a reliable and efficient means to assess invasion abilities within macrophages relative to the wild type strain. The simplicity of the method and its compatibility with standard sequencing protocols enhance its applicability across diverse laboratories, making it a significant advancement in microbiological research methodologies.