Genetic, Genomic, and System Analyses for Pure Cultures and Communities: Protocol – Ultra-High-Throughput Transposon Scanning of Bacterial Genomes

Abstract

The understanding of bacterial responses to complex environments is still a major challenge for environ- mental microbiologists. The use of next-generation sequencing technologies, such as Illumina sequencing, allows the study of complex traits from a global point of view. In this chapter, we describe a method for quantitative analysis of transposon mutant abundance in a metapopulation (transposon library) by ultra- high-throughput sequencing. The transposon library can be exposed or cultured in complex environments or conditions and compared to the starting library. Proportional abundances of individual mutants are used to infer the mutant’s fitness and hence the importance of individual gene functions in survival. The principle of the technique is based on specific amplification of transposon insertions and the directly neighboring genomic DNA, followed by sequencing of the insertion site. The protocol describes the generation of a transposon mutant library. As an example, we describe growth of the mutant library (the metapopulation) under selective conditions (here growth under salt stress and in sand). We describe how total DNA can be extracted from the metapopulations and is used to recover and sequence the insertion sites of all transposon mutants. We further describe a method of mapping the sequence reads on the reference genome and how transposon abundances can be standardized and compared across multiple conditions