Identification of spontaneous or chemically induced bacterial mutations is a powerful tool for investigation of molecular mechanisms, including the mechanism of action of novel antibiotics. However, a major bottleneck to this approach has been the identification of the causative mutation underlying a phenotype of interest. Until recently, this has required time-consuming genetic analysis. However, the advent of relatively inexpensive and rapid next-generation sequencing (NGS) technologies has revolutionized the correlation of bacterial phenotypes and genotypes. In this article we describe a simple bioinformatics pipeline to identify differences between sequenced bacterial genomes. We also describe the procedures involved in growing, extracting, and purifying DNA, and preparation of sequencing libraries for one bacterial species, Pseudomonas aeruginosa. Similar protocols will be applicable to other bacterial species. © 2018 by John Wiley & Sons, Inc.