Haplotype-resolved DiMeLo-seq maps centromeric chromatin in a complete diploid human genome.

Centromeres ensure chromosome segregation, but their chromatin organization within repetitive alpha-satellite DNA has been difficult to resolve. To address this, we generated haplotype-resolved satellite DNA annotations for the complete diploid T2T-HG002 human genome assembly, then we mapped centromere protein A (CENP-A), H3K9me3, and CpG methylation on ultra-long, adaptively sampled nanopore reads using directed methylation with long-read sequencing (DiMeLo-seq). We find that CENP-A occupies multiple discrete subdomains within hypomethylated centromere dip regions (CDRs), with constrained aggregate size and balanced CENP-A dosage between homologous chromosomes despite extensive satellite array variation. We also show that extended lymphoblastoid cell culture and induced pluripotent stem cell (iPSC) reprogramming remodel DNA methylation and alter CENP-A abundance and CDR subdomain organization. These results define a single-molecule, haplotype-resolved framework for studying human centromere plasticity, epigenetic inheritance, and chromosomal instability in development and disease.