Decoding DNA metabolism and its clinical relevance through the lens of high-throughput sequencing assays

Eukaryotic DNA metabolism, involving DNA replication and damage repair, ensures the faithful transmission of genetic information and is essential for maintaining genome integrity. Consequently, its dysregulation contributes to a broad spectrum of human diseases, including cancer and pregnancy loss. Recent advances in high-throughput sequencing (HTS) assays have enabled genome-wide, single-cell, and even single-molecule analyses of DNA metabolism dynamics within their native chromatin context, profoundly expanding our capacity to dissect these processes in vivo and to evaluate their clinical significance. In this review, we summarize HTS-based technologies that profile the entire DNA replication program, spanning initiation, elongation, termination, and replication timing, as well as the diverse pathways involved in DNA damage detection and repair. We further highlight how these approaches have been leveraged to investigate fundamental biological processes and translational applications, with particular emphasis on early embryonic development, cancer, and genome editing. Collectively, these advances illustrate how HTS has bridged molecular mechanisms with physiological and clinical insights, while pointing toward future directions including telomere-to-telomere genome analysis, single-cell multi-omics integration, and precision genomic medicine.