Next-generation sequencing (NGS) is a powerful technology that allows for high-throughput multiplex sequencing. This chapter reviews the key steps in NGS wet lab and dry lab processing. The library preparation process prepares the substrates to be sequenced through nucleic acid isolation, fragmentation, isolation, end repair, adapter ligation, size selection, and target enrichment. Several types of sequencers exist, with sequencing by ligation, synthesis, or direct analysis, and are grouped into short-read and long-read sequencing approaches. The informatics process proceeds through base calling, demultiplexing, generation of FASTQ files, sequence assembly, variant calling, variant filtering, and variant annotation. Finally, we review the flow and storage of sequencing data and the clinical applications of NGS in the care of patients.