REPLAY: A reproducible and user-friendly application for DNA replication timing analysis from Repli-seq data

Background: DNA replication timing (RT) is a fundamental feature of genome organization that is regulated in a cell-type-specific manner and frequently altered in disease. Repli-seq is the standard approach for genome-wide RT profiling; however, its analysis typically requires multiple independent tools and custom scripts, limiting reproducibility, portability, and accessibility, particularly for users without computational expertise. In addition, existing workflows often lack standardization and require substantial user intervention. Results: We developed REPLAY, a fully automated, reproducible, and user-friendly application for replication timing analysis. REPLAY is distributed as a standalone executable that enables end-to-end processing from compressed FASTQ files to genome-wide RT profiles without requiring software installation or programming experience. Through an intuitive graphical interface, users can configure analysis parameters, including input and output directories, reference genome, normalization strategy (quantile, median, or interquartile range), and smoothing. The application integrates all processing steps-quality control, trimming, alignment, binning, RT log2 calculation, normalization, smoothing, and visualization- within a single automated workflow. Application of REPLAY to publicly available datasets demonstrate accurate reconstruction of RT profiles and high reproducibility across samples. Conclusions: REPLAY offers a portable, reproducible, and accessible solution for the analysis of RT data. By eliminating the need for command-line tools and complex installations, it lowers the entry barrier enabling standardized analysis across diverse research settings.