Next-generation sequencing (NGS) technologies represented the next step in the evolution of DNA sequencing, through the generation of thousands to millions of DNA sequences in a short time. The relatively fast emergence and success of NGS in research revolutionized the ield of genomics and medical diagnosis. The traditional medicine model of diagnosis has changed to one precision medicine model, leading to a more accurate diagnosis of human diseases and allowing the selection of molecular target drugs for individual treatment. This chapter atempts to review the main features of NGS technique (concepts, data analysis, applications, advances and challenges), starting with a brief history of DNA sequencing followed by a comprehensive description of most used NGS platforms. Further topics will highlight the application of NGS towards routine practice, including variant detection, whole-exome sequencing (WES), whole-genome sequencing (WGS), custom panels (multi-gene), RNA-seq and epigenetic. The potential use of NGS in precision medicine is vast and a beter knowledge of this technique is necessary for an eicacious implementation in the clinical workplace. A centralized chapter describing the main NGS aspects in the clinic could help beginners, scientists, researchers and health care professionals, as they will be responsible for translating genomic data into genomic medicine.