The variation and evolution of complete human centromeres.

Human centromeres have been traditionally very difficult to sequence and assemble owing to their repetitive nature and large size1. As a result, patterns of human centromeric variation and models for their evolution and function remain incomplete, despite centromeres being among the most rapidly mutating regions2,3. Here, using long-read sequencing, we completely sequenced and assembled all centromeres from a second human genome and compared it to the finished reference genome4,5. We find that the two sets of centromeres show at least a 4.1-fold increase in single-nucleotide variation when compared with their unique flanks and vary up to 3-fold in size. Moreover, we find that 45.8% of centromeric sequence cannot be reliably aligned using standard methods owing to the emergence of new α-satellite higher-order repeats (HORs). DNA methylation and CENP-A chromatin immunoprecipitation experiments show that 26% of the centromeres differ in their kinetochore position by >500 kb. 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 a nearly complete turnover of α-satellite HORs, with characteristic idiosyncratic changes in α-satellite HORs for each species. Phylogenetic reconstruction of human haplotypes supports limited to no recombination between the short (p) and long (q) arms across centromeres and reveals that novel α-satellite HORs share a monophyletic origin, providing a strategy to estimate the rate of saltatory amplification and mutation of human centromeric DNA.

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.

COSMIC: Molecular Conformation Space Modeling in Internal Coordinates with an Adversarial Framework.

The fast and accurate conformation space modeling is an essential part of computational approaches for solving ligand and structure-based drug discovery problems. Recent state-of-the-art diffusion models for molecular conformation generation show promising distribution coverage and physical plausibility metrics but suffer from a slow sampling procedure. We propose a novel adversarial generative framework, COSMIC, that shows comparable generative performance but provides a time-efficient sampling and training procedure. Given a molecular graph and random noise, the generator produces a conformation in two stages. First, it constructs a conformation in a rotation and translation invariant representation─internal coordinates. In the second step, the model predicts the distances between neighboring atoms and performs a few fast optimization steps to refine the initial conformation. The proposed model considers conformation energy, achieving comparable space coverage, and diversity metrics results.

Histone Abundance Quantification via Flow Cytometry of Htb2-GFP Allows Easy Monitoring of Cell Cycle Perturbations in Living Yeast Cells, Comparable to Standard DNA Staining.

Assaying changes in the amount of DNA in single cells is a well-established method for studying the effects of various perturbations on the cell cycle. A drawback of this method is the need for a fixation procedure that does not allow for in vivo study nor simultaneous monitoring of additional parameters such as fluorescence of tagged proteins or genetically encoded indicators. In this work, we report on a method of Histone Abundance Quantification (HAQ) of live yeast harboring a GFP-tagged histone, Htb2. We show that it provides data highly congruent with DNA levels, both in Saccharomyces cerevisiae and Ogataea polymorpha yeasts. The protocol for the DNA content assay was also optimized to be suitable for both Ogataea and Saccharomyces yeasts. Using the HAQ approach, we demonstrate the expected effects on the cell cycle progression for several compounds and conditions and show usability in conjunction with additional fluorophores. Thus, our data provide a simple approach that can be utilized in a wide range of studies where the effects of various stimuli on the cell cycle need to be monitored directly in living cells.

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 α-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 α-satellite HOR array structures and four contain novel α-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 α-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 α-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 α-satellite HORs share a monophyletic origin, providing a strategy to estimate the rate of saltatory amplification and mutation of human centromeric DNA.

Complete genomic and epigenetic maps of human centromeres.

Existing human genome assemblies have almost entirely excluded repetitive sequences within and near centromeres, limiting our understanding of their organization, evolution, and functions, which include facilitating proper chromosome segregation. Now, a complete, telomere-to-telomere human genome assembly (T2T-CHM13) has enabled us to comprehensively characterize pericentromeric and centromeric repeats, which constitute 6.2% of the genome (189.9 megabases). Detailed maps of these regions revealed multimegabase structural rearrangements, including in active centromeric repeat arrays. Analysis of centromere-associated sequences uncovered a strong relationship between the position of the centromere and the evolution of the surrounding DNA through layered repeat expansions. Furthermore, comparisons of chromosome X centromeres across a diverse panel of individuals illuminated high degrees of structural, epigenetic, and sequence variation in these complex and rapidly evolving regions.