After Sanger sequencing, next-generation sequencing has emerged as a powerful tool to uncover the “submerged portion of the iceberg” in various diseases. Many rare and new mutations have been discovered using deep sequencing, which enrich our understanding in clinical genomics. Indeed, there are various high-throughput sequencing platforms available on the market, with sophisticated bioinformatic analysis to reveal the complexity of diseases and living species. We would like to call upon high quality work on this important tool to shape our current understanding and the management of various diseases. In this Special Issue, we particularly focus on how next-generation sequencing offers solutions to clinical applications, and how can this eventually provide accurate diagnosis and prescription of the right drug and the right treatment to the right patient. Existing sequencing technologies are more mature and accessible than the methodologies that are available for individual researchers to move, store, analyse and present data in a fashion that is transparent and reproducible. Here we discuss currently pressing issues with analysis, interpretation, reproducibility and accessibility of these data, and we present promising solutions and venture into potential future developments.