Infectious diseases continue to be leading causes of morbidity and mortality around the world. Hospital acquired infections are one of the major public health concerns today. This work explores how we apply next generation sequencing to understand hospital-acquired infections in the clinical setting, using advanced sequencing techniques from the research setting. In our first study, we investigated a possible Mycobacterium chelonae point outbreak of two patients in a local hospital. We performed hybrid assembly, pangenome and single nucleotide variant analysis, and found that the two strains of Mycobacterium chelonae were not genetically similar and thus were contracted from the environment separately. We defined genetic identity by establishing the divergence between genome assemblies from sequencing error through whole genome sequencing a single strain of Vibrio campbellii 39 separate times. In our second study, we investigated the association between the gut microbiome composition and bloodstream infection in critically ill patients through whole genome and metagenomic sequencing. We found that bloodstream infection species were present in the gut of half the subjects and were the dominant gut taxa in 10% of bloodstream infection episodes. Whole genome sequencing of available bloodstream infection isolates confirmed strain-level identity between gut and blood organisms in 5 of 6 episodes where the species were present in the gut. Using sequence comparison and temporal analysis, our findings suggest that the gut was a likely source of bloodstream infection in 7 of 9 episodes where the species was dominant in the gut. We concluded that translocation of dominant gut organism may contribute to bloodstream infection in some critically ill patients; thus, sequencing of the gut microbiome may provide data on the frequency and clinical correlates of bloodstream infection. Overall, our methods were able to distinguish genetic similarity between organisms both between and within subjects and shed light on the origin and dynamics of hospital-acquired infections. Our studies lay the foundations for using next generation sequencing to better understand and track the dynamics of hospital acquired infections.