3.9. Next-Generation Sequencing (NGS)

Next-generation sequencing (NGS) is a rapid and cost-effective massively parallel sequencing technology that, by focusing on the exome, is a viable alternative to whole-genome sequencing [73]. In fact, only second-generation NGS should be adopted in clinical microbiology, as it combines short-read technology for output and cost. Second-generation NGS workflows have three key phases: library preparation, template preparation, and sequencing [74]. The main difference between the different NGS platforms concerns the DNA amplification techniques used: sequencing by hybridization and sequencing by synthesis. Both platforms have been successfully applied to outbreak investigations and resistance gene mapping of several ESKAPE pathogens, including P. aeruginosa and A. baumanni [74].