Diversity and Genomic Organization of Non-B DNA Motifs in Haplotype-Resolved Human Genome Assemblies

Abstract

Long-read sequencing and telomere-to-telomere genome assemblies now enable the exploration of previously inaccessible repetitive and structurally complex regions of the human genome. Using 130 haplotype-resolved genome assemblies from 65 individuals across diverse populations, we systematically analyzed six major classes of non-B DNA motifs. By evaluating their biophysical stability, we distinguished structurally stable motifs from those forming unstable secondary structures and mapped their distribution across individuals and genomic contexts. Our work revealed significant variation at the population level in both motif abundance and predicted structural stability, uncovering previously unrecognized diversity in non-B DNA landscapes. Non-B DNA motifs exhibit notable, structure-specific enrichment in highly repetitive and evolutionarily dynamic regions that remain largely unresolved in short-read-based genomes, including centromeres, segmental duplications, structural variant breakpoints, and mobile element insertions. Our findings provide a refined view of the potential secondary-structure organization within repetitive regions of the human genome and highlight structural stability as a key factor shaping the distribution of non-B DNA motifs in regions linked to genome instability, evolution, and human variation.