Differential analysis of image-based chromatin tracing data with Dory

Spatial organization of the genome plays a vital role in defining cell identity and regulating gene expression. The three-dimensional (3D) genome structure can be measured by sequencing-based techniques such as Hi-C usually on the cell population level or by imaging-based techniques such as chromatin tracing at the single-cell level. Chromatin tracing is a multiplexed DNA fluorescence in situ hybridization (FISH)-based method that can directly map the 3D positions of genomic loci along individual chromosomes at single-molecule resolution. However, few computational tools are available for statistical differential analysis of chromatin tracing data, which are inherently high-dimensional, highly variable and contain many missing values. Here, we present Dory, a statistical method for identifying differential spatial patterns between two groups of chromatin traces. Dory quantifies pairwise spatial distances among genomic regions in a chromatin trace and applies multi-level statistical tests to detect significant structural differences between the two groups of traces. It produces a differential score matrix highlighting region pairs with significant distance difference. Applying Dory to multiple chromatin tracing datasets, we found that the detected chromatin structural changes were associated with alterations in A/B compartments and promoter-enhancer interactions correlated with differential gene expression. Dory is a robust and user-friendly computational tool for quantitative analysis of imaging-based 3D genome data that enables systematic exploration of chromatin architecture and its roles in gene regulation.