Next-generation sequencing (NGS) has revolutionized genomics by allowing rapid, high-throughput, and cost-efficient sequencing of nucleic acids, thereby providing extensive insights into genetic variation, disease origins, and evolutionary mechanisms. This chapter summaries the fundamentals of major NGS platforms, such as Illumina, PacBio, and Oxford Nanopore, and demonstrates the computational approaches necessary to handle and evaluate the huge amounts of data that are generated. The diverse applications of NGS are being explored in various fields, including clinical diagnostics where it helps to identify the pathogenic mutations and guide in personalized treatment; cancer genomics by uncovering somatic changes and molecular subtypes; and infectious disease research through the detection of antimicrobial resistance genes and pathogens. Additionally, this chapter addresses the challenges related to data storage, processing, and interpretation, highlighting the crucial role of bioinformatics tools, including sequence alignment, variant calling, and functional annotation. Future advances in technology are predicted to improve data accuracy, decrease operational costs, and broaden analytical capabilities, thus promoting ongoing innovation in both fundamental and translational genomics.