Protocol to identify higher order architectures across multiple species using HiArch

Three-dimensional genome organization plays a crucial regulatory role, but its evolutionary development across the tree of life remains unclear. Here, we present a protocol for identifying higher order genome architectures using HiArch. We describe steps for preprocessing Hi-C maps, applying NormDis normalization, and identifying global folding and checkerboard patterns. We then detail procedures for cross-sample comparison and statistical analysis. This protocol enables systematic and reproducible analysis of 3D genome organization across diverse species. For complete information on the use and execution of this protocol, please refer to Che et al. 1 1. Che, Y. ∙ Bush, S.J. ∙ Lin, H. ... The evolution of high-order genome architecture revealed from 1,000 species Cell. 2026; 189 :3760-3772.