The precise regulation of DNA replication is fundamental to maintaining genome stability and ensuring proper cell fate determination. Aberrations in DNA replication can lead to genomic instability, a hallmark of cancer and other proliferative disorders. Therefore, understanding the intricate control mechanisms governing DNA replication is crucial for developing new therapeutic strategies, including cancer treatment. Recent advancements in DNA next-generation sequencing (NGS) and state-of-the-art imaging technologies have made it possible to study DNA replication dynamics with unprecedented precision. In this project, we aim to dissect the molecular mechanisms controlling DNA replication timing and dynamics in human cells. By integrating genome-wide sequencing (here refers to nascent DNA sequencing) and super-resolution imaging, we will extract unbiased quantitative data and identify key regulatory genes and pathways involved in DNA replication in time and space .This comprehensive approach will allow us to explore the role of replication timing in maintaining genome integrity and how its dysregulation may contribute to tumorigenesis. Our research will leverage high-throughput computing resources to analyze large-scale sequencing datasets and process complex 3D super-resolution imaging data. The insights gained from this study will provide a deeper understanding of DNA replication control and lay the groundwork for developing novel cancer therapies targeting the replication machinery.