The scope of next-generation DNA sequencing (NGS) is transitioning from research to diagnostics (and beyond), but the conditions for routine clinical application have not been clearly defined. Technological limitations for sequencing a patient’s DNA fast and affordably are rapidly disappearing. At the same time, more and more is known about the role of DNA variation in disease susceptibility, disease development and response to treatment. Consequently, more and more pediatricians, cardiologists and other medical specialists would like to apply NGS-based diagnostics. The standard, comprehensive and easy-to-handle genetic test these specialists are looking for, however, is not yet available. Molecular diagnostic laboratories have started to implement NGS into their routine workflows, but are also becoming increasingly aware that the context in which they operate is changing. It becomes apparent that the major challenges are not in the technology, but rather in anticipating the changing scope and scale. Developing the infrastructure to sustainably perform NGS-based diagnostics in a changing technological, clinical and societal context is therefore more relevant than defining minimal performance criteria or standard analysis pipelines. Implementing NGS-based diagnostics comes with novel applications, emerging service models and reconfiguration of professional roles, and should thus be considered in the context of future healthcare. Here, we present the key elements for transition of NGS from research to diagnostics.