Bacterial genome sequencing has developed rapidly in the last decade and has become a primary method for analyzing the genomic basis of differences in phenotype between strains as well as being a valuable tool for public health epidemiology. This chapter provides a comprehensive workflow for bacterial genome sequencing from experimental design to data suitable for comparative genomics analysis, while mainly focusing on the challenges associated with genome assembly. This approach was successfully applied to 19 Pseudomonas aeruginosa genomes from phenotypically distinct strains.