Intraspecific sequence variation and complete haplotype-resolved assemblies refine the identification of rapidly evolving regions in humans

Humans exhibit significant phenotypic differences from other great apes, yet pinpointing the underlying genetic changes has been limited by incomplete reference genomes and a reliance on a single assembly to represent a species. We aligned 20 telomere-to-telomere (T2T) assemblies spanning great ape evolution and variation to define 1,596 consensus human ancestor quickly evolved regions (Consensus HAQERs), regions that diverged rapidly between the human-chimpanzee ancestor and an ancestral node of modern humans. Unlike prior HAQER sets based on single assemblies for a species, Consensus HAQERs incorporate population variation, reducing the likelihood of intraspecies variation appearing to be interspecies divergence. These regions contain signatures of elevated mutation rates, ancient positive selection, bivalent regulatory function, and are enriched in disease-linked loci, often emerging in previously inaccessible repetitive DNA. Through multiplex, single-cell enhancer assays, we identify HAQERs as active enhancers in the developing brain and cardiomyocytes, highlighting their potential contributions to human-specific gene regulation. Overall design: For the brain experiment, an input library of STARR-seq plasmids along with an EGFP transfection reporter plasmid are delivered to the developing mouse cerebral cortex (E14.5) via in utero electroporation. Following 48 hours of incubation, mouse brains are harvested and GFP+ cells are isolated via Fluorescent Activated Cell Sorting. Single cell sequencing is then performed to recover gene expression, EGFP transfection reporter plasmid expression, and STARR-seq reporter expression. For the heart experiment, an input library of STARR-seq plasmids along with an EGFP transfection reporter plasmid are transfected into cardiomyocyte cells differentiated from human induced pluripotent stem cells of the DU11 cell line. Following 2 days of incubation, a single cell suspension is created and GFP+ cells are isolated via Fluorescent Activated Cell Sorting. Single cell sequencing is then performed to recover gene expression, EGFP transfection reporter plasmid expression, and STARR-seq reporter expression.