Glennis A. Logsdon

Centromeres are essential chromosomal regions that ensure accurate chromosome segregation during cell division, yet their highly repetitive sequence has historically hindered their complete assembly and characterization ¹ . Consequently, the full spectrum of centromere diversity across individuals, populations and evolutionary contexts remains largely unexplored. Here we address this gap in knowledge by assembling and characterizing 2,110 centromeres from diverse individuals representing 5 continental and 28 population groups. Using bioinformatic tools tailored for centromeres, we identify variation, including 226 centromere haplotypes and 1,870 α-satellite higher-order repeat variants. While most centromeres have a single kinetochore site, we find that around 6% have di-kinetochores, and less than 1% have tri-kinetochores, which we confirm using long-read chromatin profiling and multigenerational inheritance. We also show that kinetochore position is closely associated with the underlying sequence and structure of the centromere. To understand the nature of evolutionary change, we compared these centromeres to 5,747 centromeres assembled by the Human Pangenome Reference Consortium. We show that centromeres have a 20-fold variation in mutation rate, and a subset of centromeres has evidence of archaic hominin introgression. We validate these mutation rates in a 4-generation, 28-member family and show that the kinetochore site is the most rapidly mutating region in the centromere. We propose a model that reveals an ‘arms race’ between centromeric sequence and proteins, with frequent mutations within the kinetochore site that lead to changes in genetic and epigenetic landscapes and, ultimately, rapid evolution of these critically important regions.

The human Y chromosome is among the most structurally dynamic chromosomes in the human genome, yet much of its diversity remains unresolved because of extensive palindromes, ampliconic gene families, satellite-rich heterochromatin and large segmental duplications. What remained unclear was how these diverse forms of variation fit together across the full chromosome, how often similar structures recur in different lineages, and which aspects of organization remain constrained despite rapid sequence turnover. Here, we generated and analyzed 142 nearly complete human Y chromosome assemblies from 17 major haplogroups spanning approximately 180,000 years of evolution, creating a population-scale resource for studying Y chromosome biology and diversity. These assemblies show that structural change on the Y chromosome is recurrent but constrained, even in its most repetitive regions. In the fertility-associated azoospermia factor c (AZFc) region, recurrent inversions, deletions, and complex rearrangements generate a limited repertoire of structural haplotypes. Multicopy ampliconic gene families follow distinct evolutionary paths: DAZ paralogues differ in structural constraint, RBMY evolves within a modular array, and TSPY copy number varies mainly through local expansion and contraction. The centromere and Yq12 heterochromatin vary greatly in size but retain a stable higher-order organization, including a single hypomethylated centromeric core and conserved Yq12 repeat composition and orientation. Methylation across palindromic and ampliconic regions is likewise structured by repeat class, copy order and local architecture. Together, these results provide a population-scale resource for the human Y chromosome and show that its rapid structural evolution is repeatedly funneled into a limited set of architectural outcomes.

Summary: Genomic changes are central to cancer, so sequencing is essential for understanding tumor evolution and advancing precision medicine, but resolving somatic variants and methylation is challenging in regions with repeats, reference gaps, and germline variants. ^1–4 To identify the full spectrum of somatic variants, including complex rearrangements, we construct and curate near-complete, haplotype-resolved assemblies of the most recent common ancestor of an early-passage, broadly-consented hypodiploid pancreatic cancer cell line and matched normal tissues. ⁵ The tumor assembly recapitulates all 35 chromosomes observed by karyotyping, including translocation-induced hybrid chromosomes containing dynamic dicentric and fused centromeres, nested foldback inversions causing 14 breakpoints with a haplotype switch in a single derivative chromosome, and centromeric satellite tandem duplications up to 136 kbp. Our tumor-normal assembly-based benchmark expands previous multi-technology ensemble benchmark approaches, adding >8,000 somatic small variants altering ∼40 kbp and 149 somatic SVs altering 939 kbp, including 2,764 small variants and 126 SVs not represented on GRCh38. Additionally, 5,111 somatic small variants change representation because they alter germline variants on GRCh38, impacting mutational signatures. Most source-resolved somatic LINE insertions originate from two non-reference germline LINE insertions with accessible promoter elements. These assemblies demonstrate that centromeric, acrocentric, telomeric, and other polymorphic regions, conventionally excluded from analysis, harbor extensive somatic and epigenetic changes and provide curated paired tumor-normal benchmarks to advance algorithms for repetitive regions of cancer genomes.

Centromeres ensure faithful chromosome segregation, yet how new centromeres arise and replace canonical ones remains poorly understood. Here, we investigate a polymorphic centromere repositioning event on the orangutan chromosome 10 using near-telomere-to-telomere assemblies, epigenetic profiling, and population-scale data. We identify striking heterogeneity in canonical centromeres, ranging from large, higher-order repeat α-satellite arrays to short, monomeric α-satellite tracts, alongside the emergence of neocentromeres lacking α-satellite DNA. We show a segmental duplication-mediated deletion of 3.6 Mbp that removed the higher-order repeat array, promoting centromere repositioning and neocentromere formation. Phylogenetic analyses reveal complex evolutionary dynamics, including introgression and incomplete lineage sorting in orangutan lineages. These findings demonstrate that centromere identity can evolve through structural variation and epigenetic reprogramming, highlighting its remarkable plasticity in primate genomes.

Human neocentromeres are functional centromeres demarcated by CENP-A nucleosomes that form ectopically at alpha satellite-free loci. How neocentromeres reshape local chromatin and which features of native centromeric chromatin are preserved are unknown. We generated gapless, haplotype-resolved assemblies of native and neocentromeres from three patient-derived cell lines. Integrating CpG methylation, CENP-A profiling, and single-molecule chromatin fiber sequencing, we reveal chromatin features that define the essential centromeric architecture reconstituted during neocentromere establishment. We find that a deletion within the satellite array encompassing the hypo-CpG methylation centromere dip regions (CDRs) led to native centromere inactivation, that neocentromeres harbor CDRs and a dichromatin architecture, recapitulating features of alpha-satellite centromeres, and that LINEs demarcate neocentromere boundaries, implicating transposable elements in restricting CENP-A domain spreading. Moreover, neocentromeric chromatin is incompatible with promoter-like chromatin states, redefining the regulatory landscape within genic regions. Finally, using haplotype-specific chromatin footprinting, we resolve CENP-A nucleosome chromatin architecture of active centromeres.

Centromeres are essential for accurate chromosome segregation during cell division, yet their highly repetitive sequence has historically hindered their complete assembly and characterization. Consequently, the full spectrum of centromere diversity across individuals, populations, and evolutionary contexts remains largely unexplored. Here, we address this gap in knowledge by assembling and characterizing 2,110 complete human centromeres from a diverse cohort of individuals representing 5 continental and 28 population groups. By developing a novel suite of bioinformatic tools tailored for centromeric regions, we uncover previously unknown variation within centromeres, including 226 novel centromere haplotypes and 1,870 new α-satellite higher-order repeat (HOR) variants. We find that mobile element insertions are present in 30% of centromeres, with chromosome 16 harboring Alu elements within the kinetochore site at an 11-fold higher frequency than expected. While most centromeres have a single kinetochore site, 6% of them have di-kinetochores, and <50-fold variation in mutation rate, with the most rapidly mutating centromeres on chromosome 1 and the slowest mutating centromeres on chromosome Y. Additionally, a subset of centromeres show evidence of introgression from archaic hominins, shaping their sequence, structure, and evolutionary history. We validate these centromere mutation rates in a four-generation family, spanning 28 family members and 483 accurately assembled centromeres, and show that the kinetochore site is the most rapidly mutating region in the centromere, with twofold more single-nucleotide variants than the rest of the centromeric α-satellite HOR array on average. We propose a model that reveals an 'arms race' between centromeric sequence and proteins, with frequent mutations within the site of the kinetochore that lead to changes in genetic and epigenetic landscapes and, ultimately, rapid evolution of these critically important regions.

KRAB-zinc finger proteins (KZFPs) comprise the largest family of mammalian transcription factors, rapidly evolving within and between species. Most KZFPs in human and mice have been found to repress endogenous retroviruses (ERVs) and other retrotransposons, with KZFP gene numbers correlating with the ERV load across species, suggesting coevolution. Whether new KZFPs emerge in response to ERV invasions is currently unknown. Using a combination of long-read sequencing technologies and genome assembly, we present a detailed comparative analysis of young KZFP gene clusters in the mouse lineage, which has undergone recent KZFP gene expansion and ERV infiltration. Detailed annotation of KZFP genes in a cluster on Mus musculus Chromosome 4 reveals parallel expansion and diversification of this locus in different mouse strains (C57BL/6 J, 129S1/SvImJ and CAST/EiJ) and species ( Mus spretus and Mus pahari ). Our data supports a model by which new ERV integrations within young KZFP gene clusters likely promoted recombination events leading to the emergence of new KZFPs that repress them. At the same time, ERVs also increased their numbers by duplication instead of retrotransposition alone, unraveling a new mechanism for ERV enrichment at these loci.

Human genome resequencing typically involves mapping reads to a reference genome to call variants; however, this approach suffers from both technical and reference biases, leaving many duplicated and structurally polymorphic regions of the genome unmapped. Consequently, existing variant benchmarks, generated by the same methods, fail to assess these complex regions. To address this limitation, we present a telomere-to-telomere genome benchmark that achieves near-perfect accuracy (i.e. no detectable errors) across 99.4% of the complete, diploid HG002 genome. This benchmark adds 701.4 Mb of autosomal sequence and both sex chromosomes (216.8 Mb), totaling 15.3% of the genome that was absent from prior benchmarks. We also provide a diploid annotation of genes, transposable elements, segmental duplications, and satellite repeats, including 39,144 protein-coding genes across both haplotypes. To facilitate application of the benchmark, we developed tools for measuring the accuracy of sequencing reads, phased variant call sets, and genome assemblies against a diploid reference. Genome-wide analyses show that state-of-the-art de novo assembly methods resolve 2–7% more sequence and outperform variant calling accuracy by an order of magnitude, yielding just one error per 100 kb across 99.9% of the benchmark regions. Adoption of genome-based benchmarking is expected to accelerate the development of cost-effective methods for complete genome sequencing, expanding the reach of genomic medicine to the entire genome and enabling a new era of personalized genomics.