High-throughput sequencing (HTS) technologies have revolutionized genomics, enabling the rapid and cost-effective sequencing of genomes, exomes, and transcriptomes. This review discusses the major HTS platforms, their applications, and challenges. The first human genome sequence was completed in 2001 using Sanger sequencing, which was expensive and slow. The development of HTS technologies, such as Illumina, Ion Torrent, Pacific Biosciences, and Oxford Nanopore, has dramatically reduced sequencing costs and increased throughput. Illumina's platforms, including MiSeq, NextSeq 500, and HiSeq, are widely used for high-throughput sequencing, while Pacific Biosciences and Oxford Nanopore offer long-read sequencing capabilities. Despite these advancements, challenges remain, including the difficulty of accurately characterizing large repeats, indels, and structural variations (SVs) with short-read technologies. HTS has been applied to various areas, including genome sequencing, regulatory element mapping, transcriptome analysis, and microbiome studies. It has also been instrumental in understanding rare diseases and cancer, enabling the identification of causative mutations and the discovery of new cancer drivers. However, limitations in accuracy, coverage, and the need for more comprehensive sequencing methods persist. The future of HTS in personalized medicine depends on the integration of more comprehensive techniques, such as whole-genome sequencing, and the development of standards for data interpretation. As HTS continues to evolve, it will play a crucial role in advancing our understanding of human biology and improving clinical outcomes.High-throughput sequencing (HTS) technologies have revolutionized genomics, enabling the rapid and cost-effective sequencing of genomes, exomes, and transcriptomes. This review discusses the major HTS platforms, their applications, and challenges. The first human genome sequence was completed in 2001 using Sanger sequencing, which was expensive and slow. The development of HTS technologies, such as Illumina, Ion Torrent, Pacific Biosciences, and Oxford Nanopore, has dramatically reduced sequencing costs and increased throughput. Illumina's platforms, including MiSeq, NextSeq 500, and HiSeq, are widely used for high-throughput sequencing, while Pacific Biosciences and Oxford Nanopore offer long-read sequencing capabilities. Despite these advancements, challenges remain, including the difficulty of accurately characterizing large repeats, indels, and structural variations (SVs) with short-read technologies. HTS has been applied to various areas, including genome sequencing, regulatory element mapping, transcriptome analysis, and microbiome studies. It has also been instrumental in understanding rare diseases and cancer, enabling the identification of causative mutations and the discovery of new cancer drivers. However, limitations in accuracy, coverage, and the need for more comprehensive sequencing methods persist. The future of HTS in personalized medicine depends on the integration of more comprehensive techniques, such as whole-genome sequencing, and the development of standards for data interpretation. As HTS continues to evolve, it will play a crucial role in advancing our understanding of human biology and improving clinical outcomes.