Next generation sequencing (NGS) has been vital for identifying novel pathogens and for tracing the transmission of epidemic outbreaks. NGS promises to continue to provide valuable insights for relieving or preventing infectious diseases in the future. In this thesis, NGS was applied to two projects on pathogens. The first project is based on whole genome sequencing (WGS) of Clostridium difficile. The paradoxical relationship between C.difficile and antibiotics as well as high recurrent rates have identified C. difficile infection (CDI) as a global urgent threat. However, a global perspective on the genomic and epidemiologic characteristics of this bacterium is missing to date. To fill this gap, 1501 clinical isolates were collected from 30 countries/regions across six continents to discover these patterns (Study 1). The results showed that global clinical isolates displayed five evident clades with significant continent tropism and most ribotypes have little geographic restriction when transmitted worldwide. C. difficile shared a much more conserved core genome and the pangenome was extended three times compared to previous work. Markedly different trends of antibiotic resistance among lineages (clade or ribotype) and regions (continents or countries) were identified. Hypervirulent isolates also have a high resistance rate which accounts for the rapid and widespread outbreaks. Detailed toxin polymorphism within and between major ribotypes as well as novel variants indicated that the ribotype cannot be a predictor of isolate virulence. In addition, the drug Bezlotoxumab against CDI has successfully decreased recurrence; however, it remains unknown whether benefits result from relapse (caused by the same strain) or reinfection (caused by a different strain). Therefore, the therapeutic efficacy in different recurrent patients was validated based on the genomic data (Study 2). The results showed that relapse is four times higher than reinfection and occurrs more often in high-risk populations. Bezlotoxumab could significantly reduce the cumulative incidence in relapse cases. Recurrence takes longer to develop, is more likely affected by a different strain, and outpatients have a higher hazard ratio of cumulative incidence than inpatients of relapse. The second project is based on metatranscriptomic sequencing (MGS) of wildlife bats and rodents, the ideal reservoirs of many viruses, including zoonotic viruses that have caused global viral pandemics. However, most studies describe virome diversity merely based on a few targeted viral and host species, which provides little information on the comprehensive virome landscape. In Study 3, MGS was firstly used to perform broadscale virome screening of 959 bats and 372 rodents in East Africa to fully understand the whole picture of virome.The results showed that among 268 vertebrate-infecting viruses, up to 84.7% are novel, which largely fills the evolutionary gap with different degrees among virus families. Most of these viruses are host specific, however, eight virus strains occur across different host orders/families and six of these have medium to high predicted human infection risk. Virome communities are mainly driven by host adaptation, and virome similarity declines with increasing geographic distance in specific host genera. Variation in viral richness can be explained by mammal sympatry, elevation, and human activities, highlighting that invasion of human activity and damage of wild animal habitats increase the opportunities for virus transmission to humans. In summary, the first project not only significantly enhances the available knowledge on the global landscape of evolution, distribution trends, antibiotic resistance, and toxin diversity of C. difficile, but also offers the possibility of precision therapy of recurrent CDI because of the capacity to distinguish between relapse and reinfection. Both biologists and clinicians are provided with information to potentially relieve the global burden of C. difficile infection. The second project discovered the comprehensive virome landscape in a hotspot region of zoonotic disease and evaluated the host, geographic, and ecological factors on the virome structure underlying the diversity and dispersal of viral communities. The gained fundamental knowledge is potentially instructive to better prepare for, strategize, and prevent future pandemics.