Current diagnostic methods do not sufficiently identify disease-causing pathogens in severe infectious diseasecases. Prompt identification of causative microorganisms would improve the disease outcome because of antimicrobial treatment optimization. Next-generation sequencing (NGS) is a culture-free method that analyzes theentire microbial community within a sample. Metagenomic sequencing allows us to comprehensively and quantitatively obtain the genes in all organisms present in a clinical sample. Previous studies have attempted to identifypathogens using NGS in various infectious diseases; however, there are currently no established standard protocols to identify causative microorganisms. To establish the standard NGS methods for infectious diseases, studieshave been conducted for severe infectious diseases in our laboratory: acute encephalitis and encephalopathy,acute liver failure, acute myocarditis, blood stream infection and febrile neutropenia under immunocompromisedconditions, and respiratory failure. NGS has advantages over current diagnostic methods in identifying causativepathogens in patients with severe infectious diseases. The routine diagnostic workflow using NGS is expected tobe introduced in clinical settings. NGS can potentially be used to comprehensively analyze drug resistance mutations. Because a number of drug resistance mutations have been reported in patients with cytomegalovirus (CMV)infections treated with ganciclovir, full-length antiviral gene mutation analysis was conducted in congenital CMVinfections. Moreover, NGS can reveal the background microbiome in clinical samples of infectious diseases. Recently, we investigated neonatal respiratory distress-associated microorganisms from microbial genes found inplasma and gastric fluid and assessed their relationship with clinical findings. Because NGS is a comprehensiveanalysis, potential solutions, excluding identifying causative pathogens, are expected to be found.