Biology subjects

Logsdon, G. A.

Publications and source records attributed to Logsdon, G. A..

At least 19 recordsLinked to original sources

bioRxiv2026

ZATELLITE: a toolkit to visualize and manipulate human centromeres in live cells using synthetic zinc fingers

Changes in the number of chromosomes or their spatial organization within the nucleus have critical consequences for cell fate. Yet capturing the karyotype or three-dimensional architecture of chromatin in living cells remains limited by the difficulty in labeling endogenous loci non-invasively. The most widely used tools rely on dCas9, a bulky protein whose persistent DNA binding interferes with DNA and RNA metabolism, causes DNA damage, and is hard to multiplex. We developed ZATELLITE, an AI-enabled tool to target endogenous repetitive sequences with fluorescently-tagged synthetic zinc fingers. ZATELLITE probes have key advantages over dCas9: they are smaller, easier to multiplex and, critically, they do not cause DNA damage or chromosomal abnormalities, even after long-term labeling. We generated a collection of ZATELLITE probes to label the centromeres of nearly all human chromosomes, enabling the capture of genome organization and karyotype alterations in real time in living cells. Finally, we show that ZATELLITE can be used for simultaneous labeling and epigenetic editing of centromeres. Thus, ZATELLITE is a non-toxic, versatile tool for genome visualization and manipulation.

cell biology↗

bioRxiv2026

A segmental duplication-mediated deletion leads to neocentromere formation in orangutans

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 -satellite arrays to short, monomeric -satellite tracts, alongside the emergence of neocentromeres lacking -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.

genomics↗

bioRxiv2025

Haplotype-Resolved Genomics Reveals Conserved Chromatin Architecture and Epigenetic Constraints of Human Neocentromeres

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.

genomics↗

bioRxiv2025

A global view of human centromere variation and evolution

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 -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 <<1% have tri-kinetochores, which we confirm with long-read CENP-A CUT&RUN, DiMeLo-seq, and multi-generational inheritance. We further show that the position of the kinetochore is not random and is, instead, closely associated with the underlying sequence and structure of the centromere. To understand the nature of evolutionary change, we compared 2,110 complete human centromeres to 5,747 complete centromeres recently assembled from the Human Pangenome Reference Consortium. We show that centromeres have a >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 -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.

genomics↗

bioRxiv2025

A complete diploid human genome benchmark for personalized genomics

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.

genomics↗

bioRxiv2025

Rare k-mers reveal centromere haplogroups underlying human diversity and cancer translocations

Centromeres are among the most diverse and dynamically evolving regions of the human genome and are commonly affected in various human cancers. However, organized into highly repetitive -satellite higher-order repeats (HORs), human centromere sequences have long resisted detailed genomic analysis. Although the development of long-read sequencing platforms has enabled the analysis of complete centromere sequences, their application to a large set of samples is still largely limited, preventing our understanding of centromere variation and haplotype structures across large human populations and the structural basis of centromere-involving translocations in cancer. Here we show that rare k-mers present in centromeric regions can serve as effective markers for dissecting the complexity of centromere structure, particularly that of active -satellite HOR arrays (aHOR arrays), across human populations and for understanding centromere-involving abnormalities in cancer. Based on rare k-mer-based clustering, centromere aHOR arrays are clustered into discrete haplogroups (aHOR-HGs) with distinct structural features. These k-mers were also used to develop a framework that enables the inference of haplogroups in a given sample based on short-read whole genome sequencing (WGS) data (ascairn). By applying ascairn to large-scale human population datasets (n > 3,300), we revealed the diversity of aHOR-HGs and their geographic histories across populations. The rare k-mer-based approach was also applied to investigate the structure of 1p/19q co-deletion, a highly recurrent centromere-involving translocation in IDH-mutated oligodendrogliomas. Analyzing short-read WGS data from 142 cases with 1p/19q co-deletion using rare k-mers, we showed that breakpoints of 1p/19q co-deletion were mapped to aHOR arrays in chromosomes 1 (D1Z7) and 19 (D19Z3), which was validated by long-read sequencing of two 1p/19q co-deletion-positive cases. Notably, the translocation preferentially involved haplogroups composed of haplotypes containing larger regions susceptible to rearrangement. These results highlight the role of rare k-mers in dissecting the complexity of centromere sequences and their evolutionary history as well as understanding centromere-involving abnormalities associated with human diseases.

genomics↗

bioRxiv2025

VAMPIRE: Analyzing variation and motif pattern in tandem repeats

Tandem repeats (TRs) are pervasive in eukaryotic genomes and play key roles in genome organization, evolution, and function, particularly in complex regions such as centromeres and subtelomeres. Although long-read sequencing technologies have improved the resolution of these regions, existing methods remain limited in their ability to systematically and accurately characterize large-scale TRs. Here, we introduce VAMPIRE, a k-mer-based computational tool for comprehensive TR discovery, annotation, and quantification. Unlike previous methods, VAMPIRE enables reference-free, fine-grained decomposition of both simple and complex TRs, capturing motif variation in sequence, length, and structure with high sensitivity and scalability. Applied to complete telomere-to-telomere (T2T) human and nonhuman primate (NHP) genome assemblies, VAMPIRE reveals previously unrecognized high-order repeat inversions within human centromeres--an underappreciated evolutionary mechanism contributing to centromere diversity. Additionally, the tool identifies lineage-specific and expanded TRs, including human-specific STR/VNTR expansions and NHP-specific subtelomeric heterochromatin (e.g., pCht/StSat), underscoring their dynamic turnover and structural complexity. VAMPIRE provides a robust and scalable framework for TR analysis in the era of long-read sequencing, with broad utility across human genetics, evolutionary biology, and the study of complex TRs in non-model organisms.

genomics↗

bioRxiv2025

Young KRAB-zinc finger gene clusters are highly dynamic incubators of ERV-driven genetic heterogeneity in mice

KRAB-zinc finger proteins (KZFPs) comprise the largest family of mammalian transcription factors, rapidly evolving within and between species. Most KZFPs repress endogenous retroviruses (ERVs) and other retrotransposons, with KZFP gene numbers correlating with the ERV load across species, suggesting coevolution. How 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 first 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 revealed parallel expansion and diversification of this locus in different mouse strains (C57BL/6J, 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.

genetics↗

bioRxiv2024

Identification and annotation of centromeric hypomethylated regions with Centromere Dip Region (CDR)-Finder

Centromeres are chromosomal regions historically understudied with sequencing technologies due to their repetitive nature and short-read mapping limitations. However, recent improvements in long-read sequencing allowed for the investigation of complex regions of the genome at the sequence and epigenetic levels. Here, we present Centromere Dip Region (CDR)-Finder: a tool to identify regions of hypomethylation within the centromeres of high-quality, contiguous genome assemblies. These regions are typically associated with a unique type of chromatin containing the histone H3 variant CENP-A, which marks the location of the kinetochore. CDR-Finder identifies the CDRs in large and short centromeres and generates a BED file indicating the location of the CDRs within the centromere. It also outputs a plot for visualization, validation, and downstream analysis. CDR-Finder is available at https://github.com/EichlerLab/CDR-Finder.

genomics↗

bioRxiv2024

Complex genetic variation in nearly complete human genomes

Diverse sets of complete human genomes are required to construct a pangenome reference and to understand the extent of complex structural variation. Here, we sequence 65 diverse human genomes and build 130 haplotype-resolved assemblies (130 Mbp median continuity), closing 92% of all previous assembly gaps1,2 and reaching telomere-to-telomere (T2T) status for 39% of the chromosomes. We highlight complete sequence continuity of complex loci, including the major histocompatibility complex (MHC), SMN1/SMN2, NBPF8, and AMY1/AMY2, and fully resolve 1,852 complex structural variants (SVs). In addition, we completely assemble and validate 1,246 human centromeres. We find up to 30-fold variation in -satellite high-order repeat (HOR) array length and characterize the pattern of mobile element insertions into -satellite HOR arrays. While most centromeres predict a single site of kinetochore attachment, epigenetic analysis suggests the presence of two hypomethylated regions for 7% of centromeres. Combining our data with the draft pangenome reference1 significantly enhances genotyping accuracy from short-read data, enabling whole-genome inference3 to a median quality value (QV) of 45. Using this approach, 26,115 SVs per sample are detected, substantially increasing the number of SVs now amenable to downstream disease association studies.

genomics↗

bioRxiv2024

A familial, telomere-to-telomere reference for human de novo mutation and recombination from a four-generation pedigree

Using five complementary short- and long-read sequencing technologies, we phased and assembled >95% of each diploid human genome in a four-generation, 28-member family (CEPH 1463) allowing us to systematically assess de novo mutations (DNMs) and recombination. From this family, we estimate an average of 192 DNMs per generation, including 75.5 de novo single-nucleotide variants (SNVs), 7.4 non-tandem repeat indels, 79.6 de novo indels or structural variants (SVs) originating from tandem repeats, 7.7 centromeric de novo SVs and SNVs, and 12.4 de novo Y chromosome events per generation. STRs and VNTRs are the most mutable with 32 loci exhibiting recurrent mutation through the generations. We accurately assemble 288 centromeres and six Y chromosomes across the generations, documenting de novo SVs, and demonstrate that the DNM rate varies by an order of magnitude depending on repeat content, length, and sequence identity. We show a strong paternal bias (75-81%) for all forms of germline DNM, yet we estimate that 17% of de novo SNVs are postzygotic in origin with no paternal bias. We place all this variation in the context of a high-resolution recombination map ([~]3.5 kbp breakpoint resolution). We observe a strong maternal recombination bias (1.36 maternal:paternal ratio) with a consistent reduction in the number of crossovers with increasing paternal (r=0.85) and maternal (r=0.65) age. However, we observe no correlation between meiotic crossover locations and de novo SVs, arguing against non-allelic homologous recombination as a predominant mechanism. The use of multiple orthogonal technologies, near-telomere-to-telomere phased genome assemblies, and a multi-generation family to assess transmission has created the most comprehensive, publicly available "truth set" of all classes of genomic variants. The resource can be used to test and benchmark new algorithms and technologies to understand the most fundamental processes underlying human genetic variation.

genomics↗

bioRxiv2024

Complete sequencing of ape genomes

We present haplotype-resolved reference genomes and comparative analyses of six ape species, namely: chimpanzee, bonobo, gorilla, Bornean orangutan, Sumatran orangutan, and siamang. We achieve chromosome-level contiguity with unparalleled sequence accuracy (<1 error in 500,000 base pairs), completely sequencing 215 gapless chromosomes telomere-to-telomere. We resolve challenging regions, such as the major histocompatibility complex and immunoglobulin loci, providing more in-depth evolutionary insights. Comparative analyses, including human, allow us to investigate the evolution and diversity of regions previously uncharacterized or incompletely studied without bias from mapping to the human reference. This includes newly minted gene families within lineage-specific segmental duplications, centromeric DNA, acrocentric chromosomes, and subterminal heterochromatin. This resource should serve as a definitive baseline for all future evolutionary studies of humans and our closest living ape relatives.

genomics↗

bioRxiv2024

Comparative genomics of macaques and integrated insights into genetic variation and population history

The crab-eating macaques (Macaca fascicularis) and rhesus macaques (M. mulatta) are widely studied nonhuman primates in biomedical and evolutionary research. Despite their significance, the current understanding of the complex genomic structure in macaques and the differences between species requires substantial improvement. Here, we present a complete genome assembly of a crab-eating macaque and 20 haplotype-resolved macaque assemblies to investigate the complex regions and major genomic differences between species. Segmental duplication in macaques is [~]42% lower, while centromeres are [~]3.7 times longer than those in humans. The characterization of [~]2 Mbp fixed genetic variants and [~]240 Mbp complex loci highlights potential associations with metabolic differences between the two macaque species (e.g., CYP2C76 and EHBP1L1). Additionally, hundreds of alternative splicing differences show post-transcriptional regulation divergence between these two species (e.g., PNPO). We also characterize 91 large-scale genomic differences between macaques and humans at a single-base-pair resolution and highlight their impact on gene regulation in primate evolution (e.g., FOLH1 and PIEZO2). Finally, population genetics recapitulates macaque speciation and selective sweeps, highlighting potential genetic basis of reproduction and tail phenotype differences (e.g., STAB1, SEMA3F, and HOXD13). In summary, the integrated analysis of genetic variation and population genetics in macaques greatly enhances our comprehension of lineage-specific phenotypes, adaptation, and primate evolution, thereby improving their biomedical applications in human diseases.

genomics↗

bioRxiv2024

Complete chromosome 21 centromere sequences from a Down syndrome family reveal size asymmetry and differences in kinetochore attachment

Down syndrome, the most common form of human intellectual disability, is caused by nondisjunction and chromosome 21 trisomy (T21). Small centromeres have been hypothesized to contribute to its aetiology and studies on mammals suggest that larger centromeres are more efficiently transmitted, yet complete sequencing of chromosome 21 (chr21) centromeres has been particularly challenging. Using long-read sequencing, we sequenced and assembled the centromeres from eight families that include a child with free T21 (1 trio, 6 child-mother duos, and 1 singleton) all resulting from maternal meiosis I errors. Two of these families carry the smallest chr21 centromeres (143 and 181 kbp) observed in female individuals to date, exhibiting a [~]10.7- and [~]19.4-fold centromeric -satellite higher-order repeat array size difference between the maternally inherited homologs, respectively. In both cases, the longer centromere harbors a poorly defined centromere dip region, marked by DNA hypomethylation, in the proband but not in the mother. A comparison of all proband chr21 centromeres (n=24) to those of controls (n=261) shows that small centromeres are not enriched in families with T21 (p-value=0.73); contrarily, chr21 extreme centromere size asymmetry (>10-fold) is unique of T21 (p-value=0.003), suggesting that this feature may represent a genetic risk factor for a subset of families with free T21. Additionally, phylogenetic reconstruction reveals that human chr21 has been particularly prone to such variation with some of the biggest size differences occurring over the last [~]17 thousand years of human evolution.

genomics↗

bioRxiv2023

Efficient Formation of Single-copy Human Artificial Chromosomes

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through [~]125 bp DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. Here, we describe an approach that efficiently forms single-copy HACs. It employs a [~]750 kb construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells. One-Sentence SummaryA quarter century after the first human artificial chromosomes, a solution to their uncontrolled multimerization is achieved.

molecular biology↗

bioRxiv2023

The variation and evolution of complete human centromeres

We completely sequenced and assembled all centromeres from a second human genome and used two reference sets to benchmark genetic, epigenetic, and evolutionary variation within centromeres from a diversity panel of humans and apes. We find that centromere single-nucleotide variation can increase by up to 4.1-fold relative to other genomic regions, with the caveat that up to 45.8% of centromeric sequence, on average, cannot be reliably aligned with current methods due to the emergence of new -satellite higher-order repeat (HOR) structures and two to threefold differences in the length of the centromeres. The extent to which this occurs differs depending on the chromosome and haplotype. Comparing the two sets of complete human centromeres, we find that eight harbor distinctly different -satellite HOR array structures and four contain novel -satellite HOR variants in high abundance. DNA methylation and CENP-A chromatin immunoprecipitation experiments show that 26% of the centromeres differ in their kinetochore position by at least 500 kbp--a property not readily associated with novel -satellite HORs. To understand evolutionary change, we selected six chromosomes and sequenced and assembled 31 orthologous centromeres from the common chimpanzee, orangutan, and macaque genomes. Comparative analyses reveal nearly complete turnover of -satellite HORs, but with idiosyncratic changes in structure characteristic to each species. Phylogenetic reconstruction of human haplotypes supports limited to no recombination between the p- and q-arms of human chromosomes and reveals that novel -satellite HORs share a monophyletic origin, providing a strategy to estimate the rate of saltatory amplification and mutation of human centromeric DNA.

genomics↗

bioRxiv2023

Centromere Innovations Within a Mouse Species

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying CENP-A nucleosomes at the nexus of a satellite repeat that we identified and term {pi}-satellite ({pi}-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring [~]6 Mbp of a homogenized {pi}-sat-related repeat, {pi}-satB, that contains >20,000 functional CENP-B boxes. There, CENP-B abundance drives accumulation of microtubule-binding components of the kinetochore, as well as a microtubule-destabilizing kinesin of the inner centromere. The balance of pro and anti-microtubule-binding by the new centromere permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition. TeaserChromatin and kinetochore alterations arise in response to evolutionarily rapid changes to underlying repetitive centromere DNA.

genomics↗

bioRxiv2023

Structurally divergent and recurrently mutated regions of primate genomes

To better understand the pattern of primate genome structural variation, we sequenced and assembled using multiple long-read sequencing technologies the genomes of eight nonhuman primate species, including New World monkeys (owl monkey and marmoset), Old World monkey (macaque), Asian apes (orangutan and gibbon), and African ape lineages (gorilla, bonobo, and chimpanzee). Compared to the human genome, we identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. Across 50 million years of primate evolution, we estimate that 819.47 Mbp or ~27% of the genome has been affected by SVs based on analysis of these primate lineages. We identify 1,607 structurally divergent regions (SDRs) wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (CARDs, ABCD7, OLAH) and new lineage-specific genes are generated (e.g., CKAP2, NEK5) and have become targets of rapid chromosomal diversification and positive selection (e.g., RGPDs). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species for the first time.

genomics↗