Methods for Tn-Seq Analysis in Acinetobacter baumannii.

Transposon insertion sequencing (Tn-seq) is a powerful method for identifying genes required for virtually any growth or survival trait in bacteria. The technology employs next-generation DNA sequencing to identify and quantify the relative abundances of individual transposon mutants within complex pools of such mutants. When applied to pools of thousands to millions of random transposon mutants grown under selective pressure, the technique can rapidly identify, at genome scale, the mutants and corresponding genes negatively or positively selected. This chapter presents core protocols for Tn-seq analysis of Acinetobacter baumannii: generation of a high-saturation random transposon mutant pool, and isolation and sequencing of transposon-genome junctions from such a pool for identifying and quantifying the individual mutants. With these tools, the researcher can address diverse biological questions by carrying out selective growth of a mutant pool followed by Tn-seq analysis to identify genotype-phenotype associations.