Biology subjects

Human Pangenome Reference Consortium,

Publications and source records attributed to Human Pangenome Reference Consortium,.

At least 19 recordsLinked to original sources

bioRxiv2026

The genetic architecture of human DNA methylome variation

Population DNA methylome analyses remain largely confined to reference-defined genomic space despite extensive human genetic diversity. A central challenge is therefore to define comparable methylation phenotypes across diverse genomes and map the genetic architecture of methylome variation. Here, using 460 haplotypes from the Human Pangenome Reference Consortium Release 2, we develop two complementary pangenome-based frameworks: one compares methylation within homologous regions while retaining haplotype-specific structural configuration and CpG content; the other represents CpG island (CGI) variation at homologous loci as sequence-resolved alleles. We identified 47,039 CGI alleles, including 8,040 absent from CHM13; 62% were attributable to structural variants and 31% to small variants. Through local genealogies, we reconstructed CGI gain, loss and turnover, revealing how present-day methylation differences align with CGI evolutionary trajectories. We found that genome-wide, methylation variability was buffered against genetic diversity across more than 90% of the genome, with exceptions at extreme nucleotide diversity and locally around structural variants. Our methylation framework enabled population-scale allele-aware methylation quantitative trait locus (mQTL) mapping into non-reference and structurally complex sequence, where mQTL-associated intervals were enriched. mQTLs linked methylation to transcriptomic phenotypes, population differentiation and complex traits, supporting 8% of population differentially methylated regions and colocalizing with more than half of genome-wide association study loci. Together, our study redefines human methylome analysis and our findings reveal a genome-wide architecture of genetic-epigenetic coupling, reshaping how methylation variation is interpreted across human development and disease.

genetics↗

bioRxiv2026

Pangenome-resolved polymorphic transposable elements reveal human regulatory evolution in action

Transposable elements are a major source of gene-regulatory functions. Yet this contribution is understood largely from ancient insertions already fixed in the genome: the successes, seen long after the fact. The polymorphic phase before fixation or loss remains poorly resolved because such alleles could be scored only as present or absent. We resolved segregating transposable-element alleles as sequences, epigenetic states, and transcript components using 231 haplotype-resolved human pangenomes with matched long-read multi-omics. Their origins extend beyond recent transposition; their regulatory consequences depend on methylation and transcript context and are often missed by gene-level analyses; they provide mechanisms at trait-associated loci, while selection favors element presence and absence alike. Regulatory innovation before fixation is therefore not one-way accumulation, but a mixture of gain, remodeling, and loss.

genetics↗

bioRxiv2026

Widespread structural variations at human chromosome ends

The highly repetitive regions of the human genome were long underrepresented from reference assemblies, limiting study of their biological function. Long-read sequencing and improved assembly algorithms have since resolved many of these regions, from centromeres to ribosomal DNA arrays, revealing structural variation increasingly linked to human disease. However, the subtelomeres, the repeat-rich regions adjacent to the telomeres at each chromosome end, have remained poorly characterized. Here we present a collection of complete subtelomeric sequences spanning all non-acrocentric chromosome arms, derived from 860 haploid assemblies across six ancestry groups. We find that while subtelomeres are mosaics of blocks shared between chromosome arms, individual arms diverge extensively, such that most non-acrocentric autosomal arms (54%, 21 of 39) carry multiple haplotypes differing by up to 100-200 kb. These blocks are broadly conserved across the great apes. In humans, their diversity is associated with chromosome arm rather than ancestry, suggesting that cross-arm paralogy block duplications predate human population divergence, although some haplotypes show ancestry-specific enrichment. Remarkably, these divergent haplotypes differ in gene content, driving gene copy-number variation between individuals among olfactory receptors and other genes. This study also revealed rare subtelomeric recombination. We further show that our subtelomere data set enables the accurate measurement of telomere length at individual chromosome ends from long-read data. Together, these assemblies reveal an unappreciated scale of variation at human chromosome ends and provide a resource for studying the roles of this variation in disease, telomere biology and genome evolution across diverse populations.

genomics↗

bioRxiv2026

Pangenome alignment reveals global diversity and evolution of human centromeric regions

Centromeres play essential roles in chromosome segregation and genome stability, yet they remain among the least characterized regions of the human genome. Despite advances in long-read sequencing and complete genome assembly, the extreme repetitiveness and structural complexity of these regions still challenge population-scale analysis, obscuring their mutational dynamics. The Human Pangenome Reference Consortium has now accurately assembled over 6,000 centromeres, providing an opportunity to catalog global centromere variation. However, centromeric regions have been systematically excluded from pangenome alignments due to the technical challenge of aligning their highly repetitive tandem arrays and extreme structural variability. Here we introduce Centrolign, a graph-based multiple sequence alignment tool that combines a uniqueness-driven objective function with partial-order partial-order alignment to accurately align alpha satellite higher-order repeats. By prioritizing rare matches within tandem arrays and leveraging extended centromere-spanning haplotypes formed by suppressed recombination, Centrolign produces progressive multiple sequence alignments that preserve ancestral repeat organization. Applied across human centromeres, these alignments reveal the phylogenetic structure of similar satellite array haplotypes and enable precise estimation of variation rates, structural variant frequencies, and spatial patterns of mutation within satellite arrays. Integrating Centrolign graphs with repeat annotation tools and pangenome mapping algorithms allows accurate variant calling and genotyping from long reads without prior assembly. Moreover, we show that centromere haplotypes can be accurately subtyped with k-mers alone. Together, these advances establish a robust framework for incorporating centromeres into broader pangenomes, and population genomics in general, advancing our understanding of human genome evolution and diversity.

genomics↗

bioRxiv2026

Multidimensional telomere diversity and inheritance at individual and population scales

Variation in telomere length, sequence composition and epigenetic state influences genome stability, aging and disease, yet its high-resolution characterization across species remains challenging. Here we present TeloXplorer, a computational framework for long-read data that jointly profiles telomere length, telomere variant repeats (TVRs) and DNA methylation at chromosome-end and haplotype resolution. Across simulated and empirical datasets from humans, Arabidopsis and yeast, TeloXplorer accurately resolved chromosome-end-specific telomere features and highlighted the importance of sample-matched, haplotype-resolved assemblies. Analysis of two human trios revealed concordant relative telomere-length profiles, predominantly Mendelian transmission of TVR haplotypes and family-conserved methylation patterns. Across 232 individuals from the Human Pangenome Reference Consortium, chromosome-end telomere-length rankings were conserved across five continental and 28 population groups. High-accuracy reads from 73 individuals further revealed elevated TVR haplotype diversity among individuals of African ancestry, together with extensive interchromosomal sharing and duplication of TVR architectures. Subtelomeric TAR1 elements were strongly associated with local DNA methylation and telomere motif diversity. Together, these analyses provide a multidimensional atlas of telomere diversity across species, chromosome ends, haplotypes and populations, revealing how telomere architecture varies and is inherited across biological scales.

genomics↗

bioRxiv2026

ImpuT2T: Pangenome-Based Patching for Human Genome Assemblies

With improvements in sequencing and assembly have come many high-quality telomere-to-telomere assemblies and reference pangenomes. However, the long-read sequencing recipes needed for high quality assemblies are expensive, and out of reach for many research groups. Here we propose ImpuT2T, a method that takes an assembly produced via inexpensive HiFi sequencing reads, and uses a panel of T2T (or near-T2T) assemblies to scaffold and fill ("patch") the gaps between the HiFi contigs. Benchmarking against reference assemblies demonstrates that ImpuT2T is highly effective at patching human HiFi assemblies, consistently outperforming existing patching approaches. Moreover, we show that including more haplotypes in the pangenome improves the quality of the patched assemblies, with the greatest gains achieved using the full HPRC Release 2 pangenome.

bioinformatics↗

bioRxiv2026

Concerted evolution and unorthodox recombination of human subtelomeres

Human subtelomeres contain duplicated sequence that is shared among the ends of non-homologous chromosomes and provides a substrate for ectopic exchange [1-6]. However, incomplete reference assemblies and chromosome-by-chromosome analyses have prevented a population-scale view of the extent and organization of subtelomeric exchange [7-9]. Here we apply a reference-free pangenome approach to 465 near-complete human assemblies, comparing every chromosome end against every other, and find that high-identity pseudo-homolog regions [10] occur on 41 of 48 chromosome arms. These regions group into sequence communities, sets of chromosome ends that share duplicated sequence. Across these communities, copy-number enrichment is concentrated in gene modules associated with chromosome dynamics, intracellular organization and germ-cell development. Human chromosome-contact maps show preferential proximity between subtelomeres with similar sequences, persisting even in adjacent flanks that lack the shared sequence used to define each pair. Mouse contact maps show the same preference, strongest in meiosis at the zygotene bouquet, when telomeres cluster at the nuclear envelope. In a three-generation telomere-to-telomere pedigree, whole-genome comparison identifies putative recombination between subtelomeric regions on non-homologous chromosomes from the same sequence communities, while recovering the obligate X-Y crossover in PAR1, the pseudoautosomal region shared by the X and Y chromosomes, during male meiosis. These results generalize known subtelomeric exchange systems into a near-ubiquitous architecture and support recurrent ectopic exchange as a genome-wide force in the concerted evolution of human chromosome ends.

genomics↗

bioRxiv2026

Scalable and rare-variant aware genome inference across the 1kGP cohort

Pangenome graphs built from haplotype-resolved de novo assemblies enable accurate analysis of genetic variation. The short-read-based tool PanGenie efficiently genotypes variants discovered in a pangenome across large cohorts and outper-forms linear reference-based methods for structural variants (SVs). However, it cannot detect novel variants absent from the graph, missing many rare SVs (allele frequency < 1%) and was limited to graphs with 254 haplotypes. First, we introduce a haplotype sampling step that reduces the number of haplotypes using sample-specific k-mers before genotyping, decreasing runtime twelvefold and memory usage 1.4-fold at 30x coverage. Second, we present a polishing work-flow that corrects residual errors in haplotypes inferred from PanGenie genotypes and incorporates rare and private mutations. We genotype 3,202 samples from the 1000 Genomes Project and use low-coverage ONT data (967 samples) for polishing. We achieve a median QV of 46 and provide the 1,934 polished haplotype sequences as a community resource.

bioinformatics↗

bioRxiv2026

Variation and selection at predicted G-quadruplexes across the human pangenome

G-quadruplexes (G4s), non-canonical DNA structures whose sequence motifs occupy approximately 1% of the human genome, are important for myriad cellular functions, including regulating transcription and replication. Yet they also contribute to genomic instability by increasing mutations and structural variation. Despite their significance, G4 motifs have not been studied in detail across multiple human genomes. Here, we conducted a comprehensive analysis of presence/absence and sequence variation, measured selection strength, and evaluated gene expression regulation potential for predicted G4s (pG4s) across population groups in the second release of the Human Pangenome Reference Consortium dataset, comprising high-quality, near-telomere-to-telomere diploid genomes from 231 individuals worldwide, along with three reference assemblies. Across the human pangenome, we identified over 353 million pG4s, including 1.15 million pG4s absent from reference assemblies but shared across other haplotypes. Our analysis revealed that pG4 sharing patterns recapitulate human population structure: African individuals displayed lower levels of pG4 sharing than non-Africans, whereas East Asian individuals exhibited higher levels of sharing. By analyzing the site frequency spectrum across various genomic annotations, we computed and compared selection coefficients (Sd) at pG4 vs. non-pG4 sites. As expected, the strongest purifying selection (Sd [≥] 10) was detected at protein-coding exons, where pG4 sites had similar or lower selection coefficients compared with those for pG4 sites. Strikingly, this pattern reversed at regulatory regions: although purifying selection was weaker overall at promoters, introns, enhancers, and replication origins (1 [≤] Sd < 10), pG4 sites at these regions experienced stronger selection than non-pG4 sites--suggesting that pG4s play functional roles outside coding sequences. Additionally, by integrating pG4 data with long-read transcriptome data profiles from this large cohort, we found that pG4s located at promoters and at (or near) exon-intron junctions may influence variation in gene expression levels and transcript isoforms, respectively, across the human pangenome individuals. Leveraging extensive population-scale data, our research illuminates the fundamental importance and functional relevance of G4s across human genomes.

genomics↗

bioRxiv2026

HORoSCOPE: Decoding human centromere architecture from short reads using k-mer signatures

Centromeres safeguard genome integrity, yet their roles in human disease remain understudied because of their repetitive sequence. We present HOROSCOPE, a k-mer-based framework that infers centromere architecture and generates centromere length estimates directly from short-read data. Using a reference atlas of 11,836 centromeres from telomere-to-telomere haplotypes, we derive diagnostic k-mer signatures that classify chromosome-specific architectures with 98.2% precision and 99.1% recall. Applied to 4,029 genomes from 80 populations, HOROSCOPE uncovers continental centromeric structure and African-enriched rare architectures. Across 1,359 cancer genomes, HOROSCOPE reveals a dependency of chromosomal rearrangement locations on the kinetochore attachment site position.

genomics↗

bioRxiv2026

Pansoma, a machine learning tool for identifying somatic variants using pangenome graphs

Somatic variant calling, the identification of mutations in non-germline cells acquired over an individuals lifetime, is critical for studying diseases, including cancer, and for developing precision oncology strategies. Traditional somatic variant calling methods rely on linear reference genomes, which do not adequately capture human genetic diversity and result in reference bias, compromising the accuracy of somatic variant detection. Recently developed graph-based human pangenome reference represents diverse genetic variants across human populations and has promised to drive advances in many genetics and genomics studies. In this study, we introduced Pansoma, a novel pangenome-native and machine learning-based tool specifically designed for somatic variant calling using a pangenome graph reference. Pansoma performs somatic variant detection from both short- and long-read sequencing data by learning tensor representations of alignment on graph nodes rather than on a linear reference. Pansoma outputs variant representations anchored to the pangenome graph paths and conventional somatic variant calls remapped to the linear reference. Additionally, we provide accompanying bioinformatics tools tailored for graph-based genomic data management and variant calling results analysis. Benchmarking shows that Pansoma not only improves tumor-only somatic variant detection but also preserves graph-specific variant representations that are not directly recoverable from linear- reference outputs.

genomics↗

bioRxiv2026

KaryoScope: rapid, alignment-free sequence annotation for the pangenome era

The pangenome era is producing long-read sequencing data and complete genome assemblies (1-3) at a pace that current annotation methods cannot match. Existing tools were each built for a single feature class (repeats, centromeric satellites, or genes) and falter precisely where the genome is most variable and harbours clinically important variation: the centromeres, subtelomeres, and acrocentric short arms. Here we present KaryoScope, an alignment-free method to annotate an assembly at base-pair resolution across any desired feature classes in a single pass, completing in minutes on a standard workstation. Applied to the Human Pangenome Reference Consortium Release 2 assemblies (3), KaryoScope identifies the SST1 macrosatellite as the recurrent sequence at Robertsonian translocation fusion points (4, 5), delivers the first pangenome-wide census of D4Z4 macrosatellite structural diversity at the 4q and 10q subtelomeres relevant to facioscapulohumeral muscular dystrophy (6), and reveals previously uncharacterised centromere structural polymorphism, including chromosome-specific satellite loss and megabase-scale rearrangement validated by fluorescence in situ hybridization. A pre-built KaryoScope database for the human genome is distributed alongside the tool, and additional databases can be built for any reference genome or annotation source. Together, these capabilities bring the most variable regions of the genome within reach for comparative, clinical, and pangenome-scale analysis. KaryoScope is available at https://github.com/barthel-lab/KaryoScope.

bioinformatics↗

bioRxiv2026

Single-molecule variation in telomeric sequence and structure across humans

The repetitive architectures of telomeric and subtelomeric regions have obscured studies of their genetic variation and chromatin organization across the human population. Here, we integrate near-complete diploid genome assemblies from 212 individuals with matched long-read sequencing data to construct an atlas of 316,146 telomere-spanning molecules across 12,080 chromosome-end-resolved telomere arrays. This atlas reveals that nearly every chromosome end harbors a structured and unique pattern of telomere variant repeats (TVR), or TVR code, with subtelomere-proximal TVR codes being heritable, somatically stable, and influenced by subtelomeric TAR1 regulatory elements. Despite ongoing cycles of telomere shortening and elongation in the germline, proximal TVR codes are maintained across the human population. These TVR codes expose rare telomerase-independent events that lengthen telomeres in the germline, including interchromosomal telomere exchange and recurrent internal duplications within telomere arrays. Furthermore, single-molecule chromatin fiber sequencing across 26,972 molecules spanning the telomere-subtelomere boundary confirms that TVR-rich regions adopt telomeric chromatin but introduce discrete discontinuities into otherwise compact telomeric chromatin fibers. Together, our results link chromosome-end sequence variation to telomere cap formation and telomerase-independent telomere extension mechanisms in the human germline.

genomics↗

bioRxiv2026

Biobank-scale genotyping of Robertsonian translocations reveals hidden structural variation on the human acrocentric chromosomes

Balanced Robertsonian translocation (ROB) is the most common chromosomal rearrangement in humans, with an estimated occurrence of 1 in 800 in newborn studies. Carriers are at increased risk of cancer and often diagnosed at fertility clinics after facing recurrent miscarriages, infertility, or aneuploid offspring. Genotyping carriers with DNA sequencing has been challenging because of gaps and misrepresentation of the translocation fusion site in the human reference genome. Only recently, telomere-to-telomere (T2T) human genomes successfully revealed sequences of the acrocentric short arms, including the most common ROB fusion site. A ROB results in loss of two ribosomal DNA (rDNA) arrays and its adjacent distal sequences, including the highly conserved distal junction (DJ). Here, we present a novel method to type ROB carriers directly from short sequencing reads by estimating DJ copy number. We demonstrate that our method successfully genotypes ROBs using a reference-free approach or alignments to either T2T-CHM13v2 or GRCh38. Applying the method to a cohort of healthy newborns and family members (n=4,172) as well as the UK Biobank (n=490,416), we find candidate ROBs at a frequency consistent with the previously reported 1 in 800 incidence (0.11-0.12%). In addition to ROB carriers, we report the frequency of one DJ loss (9, 2.8-3.4%) or gain (11+, 8.4-9.3%) from the two cohorts and the 1000 Genomes Project (n=3,202), and characterize the underlying structural variation in near-T2T genome assemblies from the Human Pangenome Reference Consortium. Importantly, our method provides the first sequencing-based diagnostic for Robertsonian chromosomes and can be applied to low-coverage sequencing data, enhancing its clinical applicability and enabling new studies of structural variation on the acrocentric chromosomes.

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

SNP calling, haplotype phasing and allele-specific analysis with long RNA-seq reads

Long-read RNA sequencing (lrRNA-seq) is a powerful technology to link transcript structures to genetic variants but such analysis is not often performed due to the lack of end-user tools. Here, we introduce longcallR for joint SNP calling, haplotype phasing, and allele-specific analysis, which achieves high accuracy on benchmark datasets. Applied to 202 human samples, longcallR identified 88 significant allele-specific splicing events per sample on average. 46% of them involved unannotated junctions.

bioinformatics↗

bioRxiv2022

Pangenome Graph Construction from Genome Alignment with Minigraph-Cactus

Reference genomes provide mapping targets and coordinate systems but introduce biases when samples under study diverge sufficiently from them. Pangenome references seek to address this by storing a representative set of diverse haplotypes and their alignment, usually as a graph. Alternate alleles determined by variant callers can be used to construct pangenome graphs, but thanks to advances in long-read sequencing, high-quality phased assemblies are becoming widely available. Constructing a pangenome graph directly from assemblies, as opposed to variant calls, leverages the graphs ability to consistently represent variation at different scales and reduces biases introduced by reference-based variant calls. Pangenome construction in this way is equivalent to multiple genome alignment. Here we present the Minigraph-Cactus pangenome pipeline, a method to create pangenomes directly from whole-genome alignments, and demonstrate its ability to scale to 90 human haplotypes from the Human Pangenome Reference Consortium (HPRC). This tool was designed to build graphs containing all forms of genetic variation while still being practical for use with current mapping and genotyping tools. We show that this graph is useful both for studying variation within the input haplotypes, but also as a basis for achieving state of the art performance in short and long read mapping, small variant calling and structural variant genotyping. We further measure the effect of the quality and completeness of reference genomes used for analysis within the pangenomes, and show that using the CHM13 reference from the Telomere-to-Telomere Consortium improves the accuracy of our methods, even after projecting back to GRCh38. We also demonstrate that our method can apply to nonhuman data by showing improved mapping and variant detection sensitivity with a Drosophila melanogaster pangenome.

bioinformatics↗

bioRxiv2022

Recombination between heterologous human acrocentric chromosomes

The short arms of the human acrocentric chromosomes 13, 14, 15, 21, and 22 share large homologous regions, including the ribosomal DNA repeats and extended segmental duplications (Floutsakou et al. 2013; van Sluis et al. 2019). While the complete assembly of these regions in the Telomere-to-Telomere consortiums CHM13 provided a model of their homology (Nurk et al. 2022), it remained unclear if these patterns were ancestral or maintained by ongoing recombination exchange. Here, we show that acrocentric chromosomes contain pseudo-homologous regions (PHRs) indicative of recombination between non-homologs. Considering an all-to-all comparison of the high-quality human pangenome from the Human Pangenome Reference Consortium (HPRC) (Liao et al. 2022), we find that contigs from all of the acrocentric short arms form a community similar to those formed by single chromosomes or the sex chromosome pair. A variation graph (Garrison et al. 2018) constructed from centromere-spanning acrocentric contigs indicates the presence of regions where most contigs appear nearly identical between heterologous CHM13 acrocentrics. Except on chromosome 15, we observe faster decay of linkage disequilibrium in the PHRs than in the corresponding short and long arms, indicating higher rates of recombination (N. Li and Stephens 2003; Huttley et al. 1999). The PHRs include sequences previously shown to lie at the breakpoint of Robertsonian translocations (Jarmuz-Szymczak et al. 2014), and we show that their arrangement is compatible with crossover in inverted duplications on chromosomes 13, 14, and 21. The ubiquity of signals of recombination between heterologous chromosomes seen in the HPRC draft pangenomes acrocentric assemblies suggests that these shared sequences form the basis for recurrent Robertsonian translocations, providing sequence and population-based confirmation of hypotheses first developed cytogenetically fifty years ago (Hamerton et al. 1975).

genomics↗