Accurate and robust 3D genome feature discovery from multiplexed DNA FISH

Multiplexed DNA FISH (also known as FISH Omics) is a powerful imaging technology to map three-dimensional (3D) genome organization in thousands of single cells at kilobase resolution. However, existing computational approaches to identify 3D genome features, such as A/B compartments, topologically associating domains (TADs) and chromatin loops, have been mainly adapted from Hi-C and do not account for the distinct measurement errors across the three XYZ physical axes. Here we present ArcFISH (accurate and robust chromatin analysis from FISH data), which explicitly models axis-specific measurement errors in a unified statistical framework to detect multi-level 3D genome features. Comprehensive evaluations using multiple simulated as well as experimental datasets show that ArcFISH reliably identifies chromatin loops under heterogeneous measurement errors, detects TAD boundaries highly enriched for active histone marks H3K4me3 and H3K36me3, and requires as few as 30 chromatin traces to faithfully assign A/B compartments. Applying ArcFISH to multiplexed DNA FISH data from mouse embryonic stem cells and mouse excitatory neurons reveals cell-type-specific chromatin loops and TADs, overlapping with differentially expressed genes. ArcFISH is freely available at https://hyuyu104.github.io/ArcFISH.