The Use of Tn-Seq and the FiTnEss Analysis to Define the Core Essential Genome of Pseudomonas aeruginosa.

Transposon-insertion sequencing (Tn-Seq) allows for identification of bacterial genes and pathways essential for growth under a given condition. A transposon mutant is created by the stable and random integration of a transposable element into a genome of interest, followed by a period of outgrowth and selection for relative fitness on one or more growth media. By pooling hundreds of thousands of mutants, sequencing the transposon-genomic DNA junctions, and mapping sequencing reads to the genome, one can identify an abundance of reads in nonessential insertion regions and the absence of reads in essential regions and thus identify which genes are essential for a given growth condition. By performing this method iteratively across multiple strains and growth conditions, one can define a core essential genome for a species. Here, we describe this methodology in detail and its application for the species Pseudomonas aeruginosa, from generating mutants to the analysis of nonessential versus essential genes using the freely available software "FiTnEss".