Transposon Insertion Site Sequencing in a Urinary Tract Model

Transposon sequencing (Tn-seq) is a technique that combines quantitative next-generation sequencing and a saturating transposon mutant library for an organism of interest, and ultimately allows for quantitation of the relative abundance of all of the mutants under a given condition, such as during experimental infection. The massively parallel sequencing capabilities of this technique provide a significant advance over more traditional methods of screening transposon mutant pools or individually determining the fitness contribution of genes of interest. Here, we describe a method for generating a genome-saturating transposon mutant library in Proteus mirabilis, determining the appropriate number of mutants for inoculation in an experimental infection model, preparing transposon insertion junctions for Illumina sequencing, and downstream analysis of mapped DNA sequencing reads for estimation of the contribution of each gene in the genome to fitness during infection.