Whole genome sequencing (WGS) has become the new gold standard for typing and characterization of pathogens. WGS enables understanding transmission chains of bacteria, exchange of mobile genetic elements, and variation in viruses with the highest resolution. In the near future, national and international databases will allow the tracking of pathogen transmissions based on WGS data in and across complex settings such as humans, animals, food, water and other environmental sources. Understanding the complexity and dynamics of and across these compartments will also help us to reduce transmissions of multi-drug resistant pathogens. Several important challenges for Public Health and One Health-related questions need to be identified such as defining standards for data analysis, accessing important epidemiological metadata, sharing data sharing while respecting data protection and ethical considerations. This symposium brought together leading institutions and fostered the ongoing discussion on the identified challenges, paving the way for solutions in public health, diagnostics, and research. This symposium report summarizes the key messages.