Abstract
G-quadruplex (G4) ChIP-Seq data are critical for studying the roles of G4 structures in various biological processes, yet their reproducibility remains systematically uncharacterized. In this study, we evaluated the consistency of in vivo G4 peaks across multiple replicates in three publicly available datasets. We observed considerable heterogeneity in peak calls, with only a minority of peaks shared across all replicates. To address this challenge, we compared three computational methods-IDR, MSPC, and ChIP-R-for assessing reproducibility and found that MSPC is the optimal solution in reconciling inconsistent signals in G4 ChIP-Seq data. We further demonstrated that employing at least three replicates significantly improved detection accuracy compared to conventional two-replicate designs, while four replicates proved sufficient to achieve reproducible outcomes, with diminishing returns beyond this number. Moreover, we showed that the reproducibility-aware analytical strategies can partially mitigate the adverse effects of low sequencing depth, though they do not fully substitute for high-quality data. Based on our findings, we recommend 10 million mapped reads as a minimum standard for G4 ChIP-Seq experiments, with 15 million or more reads being preferable for optimal results. Our study provides practical guidelines for experimental design and data analysis in G4 studies, emphasizing the importance of replication and robust bioinformatic strategies to enhance the reliability of genome-wide G4 mapping.