Haplotype-resolved genome architecture mapping uncovers pervasive structural heterogeneity between human homologous chromosomes

Resolving the three-dimensional structure of chromatin with haplotype specificity remains a fundamental challenge for understanding genetic and epigenetic contributions to gene regulation in healthy development and in disease. Phasing of chromatin contacts derived by ligation-dependent methods relies on high single-nucleotide variant (SNV) density, which limits its applicability in human genomes where SNVs are sparse. Here, we present CoPhasing, a strategy that leverages the intrinsic haplotype fidelity of Genome Architecture Mapping (GAM), a ligation-free method that inherently captures haplotype-matched genomic neighborhoods in thin nuclear slices. CoPhasing assigns SNV-free reads to their haplotype through proximity to informative reads, enabling efficient and accurate phasing even in SNV-sparse regions. Applying CoPhasing genome-wide reveals extensive, previously inaccessible structural heterogeneity between human homologous chromosomes, demonstrating the power of GAM CoPhasing to investigate allele-specific differences in 3D genome organization and their relevance to gene regulation and disease.