Next-generation sequencing (NGS) technologies generate higher resolution and less noise data that can allow the assembly of bacterial genome sequences. It also enables the characterization and quantification of transcriptomes, and the genome-wide profiling of DNA-protein interactions. With decreasing cost of NGS, such revolutionary advances in technology has become a powerful tool for studying bacterial genome complexity, which in turn will be used to design synthetic genome. This review describes the NGS approaches, the challenges associated with their application and the advances made so far in characterizing bacterial genomes, transcriptomes, and interactomes. We anticipate these high-throughput data to be a resourceful and broadly used for elucidating bacterial cells at the system level and furthermore, for the synthesis of intelligent biological systems for biotechnological purposes.