Biology subjects

Ranallo-Benavidez, T. R.

Publications and source records attributed to Ranallo-Benavidez, T. R..

11 recordsLinked to original sources

bioRxiv2026

HPRC2: A human pangenome reference with near-complete coverage of common genetic variation

A pangenome reference overcomes the inherent limitation of any individual reference genome by integrating the variation present in a population. We present the Human Pangenome Reference Consortiums (HPRC) Release 2 (HPRC2), an openly available, second phase pangenome that is an approximately fivefold expansion in genome number over HPRC Release 1 (HPRC1) and measurable improvement in genome completeness, contiguity, and accuracy. Selecting samples with a principled algorithm prioritising common variant coverage, HPRC2 contributes 460 haplotypes that together capture over 99% of common variation observed in the All of Us Research Program v8 cohort. Combining high-coverage long and ultra-long reads with modern assemblers and polishers, we produce thousands of telomere-to-telomere (T2T) chromosomes, and relative to HPRC1 halve the number of structurally unreliable regions as well as individual base errors per haplotype. We complement the assemblies with whole genome multiple alignments and gene annotations, and derive formal pangenome coordinate systems for addressing off-reference variation, demonstrating that individual human genomes contain more than one hundred thousand variants not succinctly described with respect to existing reference genomes. We also present the first matched long-read backed pantranscriptome and panepigenome at this scale, provide continuous local-ancestry estimates spanning every genome, and outline a host of new tools and applications that leverage the pangenome resource for improved genomics analysis.

genomics↗

bioRxiv2026

Telomere Dysfunction in Human Astrocytes Drives Acrocentric Chromosome Instability and Nucleolar Reorganization

Loss of p53 and Rb enables continued cell division despite progressive telomere erosion, ultimately triggering telomere crisis. While recent work has clarified mechanisms of chromatin bridge resolution, the longitudinal dynamics of structural damage through crisis remain incompletely understood. We transduced normal human astrocyte (NHA) cells with HPV18 E6/E7 and tracked them across extended population doublings. NHA E6/E7 cells showed progressive telomere shortening, anaphase bridges, and a growth plateau consistent with crisis. Multicolor FISH revealed subclonal chromosomal abnormalities largely invisible to short-read sequencing, with acrocentric chromosomes disproportionately affected: chromosome 13 was abnormal in over 92% of metaphases, and chromosomes 21 and 22 were similarly enriched. Translocations involving chromosome 13 were transient, replaced at later passages, while numerical aberrations persisted. Immunofluorescence revealed compact spherical nucleoli replaced by dispersed necklace-like structures, indicating a large-scale reorganization of nucleolar structure in response to telomere dysfunction. To determine whether these changes reflected altered chromosomal organization in the nucleus, we performed Hi-C and deployed KaryoScope, our alignment-free k-mer-based approach that recovers trans-chromosomal signal from repetitive acrocentric short arms. Inter-chromosomal contacts among nucleolar organizing region-bearing acrocentric chromosomes were markedly and persistently depleted in E6/E7 cells. Together, cytogenetic, imaging, and chromatin-contact data identify the nucleolus as a structural nexus linking telomere dysfunction to large-scale genomic rearrangement.

cancer biology↗

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

A complete human pancreatic cancer genome

Cancer genome sequencing is essential for understanding tumor evolution and advancing precision medicine.1 However, reference gaps and germline variants obscure detection of small and large somatic variants and methylation in repetitive regions.1-3 It is common for tumor cells to gain or lose chromosome arms due to somatic structural changes that occur inside highly repetitive satellite DNA sequences in the centromeres.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. The tumor assembly completely recapitulates all 35 tumor chromosomes observed with karyotyping, with multiple translocation-induced hybrid chromosomes. The hybrid chromosomes contain putative functional dicentric and fused centromeres, nested foldback inversions causing 14 breakpoints with a haplotype switch in a single event, and centromeric satellite tandem duplications up to 136 kbp. Direct comparison of tumor and normal assembly haplotypes uncovers >7,000 variants altering >1 Mbp of sequence in repetitive regions that have been hidden by reference gaps and germline variants. 44 % of somatic small variants change representation because they alter germline variants on GRCh38, impacting mutational signatures and kataegis/omikli clusters. Most somatic LINE insertions originate from two hypomethylated non-reference germline LINE insertions, highlighting their impact on insertion mutation burden. These assemblies demonstrate that centromeric, acrocentric, and telomeric regions conventionally excluded from analysis harbor extensive somatic and epigenetic changes. Resolving complete tumor genomes enables a deeper understanding of cancer structural plasticity and the endpoints of breakage-fusion-bridge cycles. These assembled, curated paired normal-tumor benchmarks will serve as a critical foundation for developing future algorithms to characterize the most intractable regions of cancer genomes.

genomics↗

bioRxiv2026

Chromosome-arm-specific telomere length governs dual modes of structural genome evolution in IDH-mutant astrocytoma

IDH-mutant astrocytomas maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, producing extreme inter-arm telomere length heterogeneity, yet how this heterogeneity shapes structural genome evolution remains unknown. Using Oxford Nanopore long-read sequencing of 20 IDH-mutant astrocytomas, we profiled structural variants (SVs), copy number variants, extrachromosomal DNA (ecDNA) and measured allele-specific telomere lengths from individual long reads. We identified pervasive complex rearrangements, including chromothripsis and foldback events consistent with breakage-fusion-bridge cycles, and widespread ecDNAs. SV breakpoints were enriched at telomeric and centromeric regions regardless of local telomere length, revealing constitutive structural fragility. Arm-level telomere length analysis uncovered a dual-mode model: arms with short telomeres preferentially harbored breakage-associated events, while arms with long ALT-maintained telomeres were enriched for ecDNA and amplification-associated events. These findings identify chromosome-arm-specific telomere length as a determinant of structural genome evolution in ALT-driven tumors.

cancer biology↗

bioRxiv2026

Hierarchical genomic feature annotation with variable-length queries

K-mer-based methods are widely used for sequence classification in metagenomics, pangenomics, and RNA-seq analysis, but existing tools face important limitations: they typically require a fixed k-mer length chosen at index construction time, handle multi-matching k-mers (whose origin in the indexed data is ambiguous) in ad-hoc ways, and some resort to lossy approximations, complicating interpretation. We present HKS, a data structure for exact hierarchical variable-length k-mer annotation. Building on the Spectral Burrows- Wheeler Transform (SBWT), a single HKS index is constructed for a specified maximum query length s, and supports queries at any length k [≤] s. HKS associates each k-mer with exactly one label from a user-defined category hierarchy, where multi-matching k-mers are resolved to their most specific common node in the hierarchy. We formalize a feature assignment framework that partitions indexed k-mers into disjoint sets according to a user-defined category hierarchy. To recover specificity lost to multi-matching and novel k-mers, we introduce a hierarchy-aware smoothing algorithm that makes use of flanking sequence context. We validate the approach by assigning each query k-mer to a specific chromosome across human genome assemblies, including the T2T-CHM13v2.0 reference as a positive control and two diploid genomes of different ancestries (HG002, NA19185). Smoothing increases overall concordance from [~]81% to [~]97%, with residual errors attributable to known biological phenomena including acrocentric short-arm recombination and subtelomeric duplications. In performance benchmarks against Kraken2, HKS provides comparable query throughput while providing exact, lossless annotation across all k-mer lengths simultaneously from a single index. A prototype implementation is available at https://github.com/jnalanko/HKS.

bioinformatics↗

bioRxiv2025

Mapping the Telomeric 3D Interactome with Telomere-C Reveals Repetitive Element Hubs Associated with Telomere Maintenance

Telomeres are essential for genome integrity, but the accurate, high-resolution mapping of their three-dimensional (3D) chromatin interactions, a process thought to mediate gene regulation and telomere maintenance, has been limited by the repetitive nature of subtelomeric DNA and shortcomings of traditional sequencing-based chromatin conformation capture methodologies. To overcome these challenges, we developed Telomere Conformation Capture by sequencing (Telomere-C), a novel high-throughput approach for the genome-wide identification of telomeric chromatin interactions. We applied Telomere-C to three normal fibroblast cell lines (BJ, IMR90, WI38), one telomerase-positive cancer cell line (HeLa S3) and two cancer cell lines (U2-OS and WI38-VA13) that maintain telomeres by alternative lengthening of telomeres (ALT). We generated millions of telomere-associated reads per sample and identified up to 41,026 interaction peaks. We successfully validated previously-described telomeric interaction sites (e.g., TERT and DUX4), and critically, revealed a novel class of ultra-long-range interactions extending over 5 Mb away from the telomere, which surprisingly constitute over 80% of all identified contacts. Contrary to prior studies focusing on protein coding gene interactions, we discovered that the telomeric 3D interactome is overwhelmingly anchored at repetitive element hubs, particularly interstitial telomeric sequence (ITS), telomere-associated repeat 1 (TAR1) and D20S16 elements. We found that the clustering of these interactions correlates strongly with cell-type-specific telomere maintenance. Most notably, D20S16 telomeric interactions were uniquely and highly enriched in ALT cancer cells, suggesting a mechanistic link. Taken together, our study effectively constructs the first high resolution maps of the telomeric 3D interactome, redefining its scope to be dominated by ultra-long-range contacts with repetitive elements. This work provides fundamental insights into telomeric nuclear organization and establishes the telomeric D20S16 interaction as a molecular signature for the ALT pathway.

genomics↗

bioRxiv2025

Diploid genome assembly of human fibroblast cell lines enables clone specific variant calling, improved read mapping and accurate phasing

Human cell lines are fundamental tools in biomedical research and are widely used in disease modeling, drug development, and many other domains. Here, we present chromosome-level, phased diploid genome assemblies of two popular human cell lines: the BJ foreskin fibroblast line and the IMR-90 fetal lung fibroblast line. Our high-quality assemblies, generated using long-read and Hi-C sequencing data, reveal substantial structural variation, including more than 50,000 insertions, deletions, duplications, and inversions compared to the recent T2T-CHM13v2.0 reference. Our assemblies provide detailed maps of genetic variation, enabling more accurate variant calling and the ability to phase reads when using newly generated or historical sequencing data on these cell lines or their derivatives. All assemblies and associated data have been made available as a resource for the research community. We envision that diploid genome assembly will become a cornerstone approach for personalized medicine in the near future.

genomics↗

bioRxiv2020

SVCollector: Optimized sample selection for cost-efficient long-read population sequencing

An increasingly important scenario in population genetics is when a large cohort has been genotyped using a low-resolution approach (e.g. microarrays, exome capture, short-read WGS), from which a few individuals are selected for resequencing using a more comprehensive approach, especially long-read sequencing. The subset of individuals selected should ensure that the captured genetic diversity is fully representative and includes variants across all subpopulations. For example, human variation has historically been focused on individuals with European ancestry, but this represents a small fraction of the overall diversity. To address this goal, SVCollector (https://github.com/fritzsedlazeck/SVCollector) identifies the optimal subset of individuals for resequencing. SVCollector analyzes a population-level VCF file from a low resolution genotyping study. It then computes a ranked list of samples that maximizes the total number of variants present from a subset of a given size. To solve this optimization problem, SVCollector implements a fast greedy heuristic and an exact algorithm using integer linear programming. We apply SVCollector on simulated data, 2504 human genomes from the 1000 Genomes Project, and 3024 genomes from the 3K Rice Genomes Project and show the rankings it computes are more representative than widely used naive strategies. Notably, we show that when selecting an optimal subset of 100 samples in these two cohorts, SV-Collector identifies individuals from every subpopulation while naive methods yield an unbalanced selection. Finally, we show the number of variants present in cohorts of different sizes selected using this approach follows a power-law distribution that is naturally related to the population genetic concept of the allele frequency spectrum, allowing us to estimate the diversity present with increasing numbers of samples.

bioinformatics↗

bioRxiv2019

GenomeScope 2.0 and Smudgeplots: Reference-free profiling of polyploid genomes

An important assessment prior to genome assembly and related analyses is genome profiling, where the k-mer frequencies within raw sequencing reads are analyzed to estimate major genome characteristics such as genome size, heterozygosity, and repetitiveness. Here we introduce GenomeScope 2.0 (https://github.com/tbenavi1/genomescope2.0), which applies combinatorial theory to establish a detailed mathematical model of how k-mer frequencies are distributed in heterozygous and polyploid genomes. We describe and evaluate a practical implementation of the polyploid-aware mixture model that, within seconds, accurately infers genome properties across thousands of simulated and eleven real datasets spanning a broad range of complexity. We also present a new method called Smudgeplots (https://github.com/KamilSJaron/smudgeplot) to visualize and infer the ploidy and genome structure of a genome by analyzing heterozygous k-mer pairs. We successfully apply the approach to systems of known variable ploidy levels in the Meloidogyne genus and also the extreme case of octoploid Fragaria x ananassa.

bioinformatics↗

bioRxiv2018

Genomic features of asexual animals

Evolution without sex is predicted to impact genomes in numerous ways. Case studies of individual parthenogenetic animals have reported peculiar genomic features which were suggested to be caused by their mode of reproduction, including high heterozygosity, a high abundance of horizontally acquired genes, a low transposable element load, or the presence of palindromes. We systematically characterized these genomic features in published genomes of 26 parthenogenetic animals representing at least 18 independent transitions to asexuality. Surprisingly, not a single feature was systematically replicated across a majority of these transitions, suggesting that previously reported patterns were lineage specific rather than illustrating general consequences of parthenogenesis. We found that only parthenogens of hybrid origin were characterized by high heterozygosity levels. Parthenogens that were not of hybrid origin appeared to be largely homozygous, independently of the cellular mechanism underlying parthenogenesis. Overall, despite the importance of recombination rate variation for the evolution of sexual animal genomes, the genome-wide absence of recombination does not appear to have had the dramatic effects which are expected from classical theoretical models. The reasons for this are probably a combination of lineage-specific patterns, impact of the origin of parthenogenesis, and a survivorship bias of parthenogenetic lineages.

genomics↗