Since the application of modern technology in medicine, scientists always tried to understand the real nature of nucleic acids. To this end, a great innovation was brought by the pioneer work of Sanger et al. in the late 70s, when they elaborated the most used method to sequence DNA [1], still considered as the gold standard in molecular diagnostics, even though it is expensive and time-consuming. Growing efforts have been made to widen technical knowledge in this field until the discovery of the “second and third-generation sequencing” methodsThey are both part of the next-generation sequencing (NGS) technology, a group of techniques revolutionizing the standard concept of nucleic acids sequencing. The great success of NGS technology is due to the capability of massively sequencing millions of DNA reads, with the possibility to perform, at least, multi-gene analysis, by using very low amount of nucleic acids. NGS technology is suitable for rapid and efficient sequencing of complex genomes too, with consequent time and cost reduction. Furthermore, it can also count on a noteworthy flexibility: in fact, its application has been reported to be successful in different research fields such as molecular diagnostics of genetic diseases, infectious diseases, cancer and pharmacogenomics