Biology subjects

Guarracino, A.

Publications and source records attributed to Guarracino, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Measuring satellite arrays of the human Y chromosome from sequencing reads

Human Y chromosome satellites vary widely but remain difficult to measure in large cohorts. In response, we developed Yruler, which uses pangenome assemblies to measure satellite lengths, composition and Y dosage from sequencing reads without alignment. Analyses reveal centromere-length differences between related lineages, extend observations of short non-centromeric arrays across a lineage, and show unequal component variability and composition changes with heterochromatin length within lineages. Yruler brings satellite variation into population-scale phenotype studies.

genomics↗

bioRxiv2026

Somatic variant detection using an individual-specific pangenome reference: a case study of a pediatric acute myeloid leukemia patient

Background: Reference bias can cause false or missed somatic variant calls, also in clinically relevant genes. Pangenomes aim to reduce this bias, but evidence of their diagnostic benefit is lacking. We tested how an individual-specific pangenome affects somatic variant calling in bulk samples and single cells. Results: We built an individual-specific pangenome from Nanopore assemblies of normal hematopoietic stem and progenitor cells of a child with acute myeloid leukemia. We aligned short-read whole-genome sequencing data of bulk and single-cell samples to hg38 and to the pangenome, and called somatic variants with standard downstream tools after anchoring pangenome alignments to hg38. Single nucleotide variants were mostly shared, while indel calls unique to the pangenome were fewer than those unique to hg38, mostly in repeats. Both approaches detected NUP98-NSD1 and FLT3 internal tandem duplication (FLT3-ITD) drivers. The pangenome-based phylogeny showed more parallel branching in comparison to hg38-based tree. The pangenome removed three false-positive PRSS1 variants and, in single cells, frameshift insertion calls next to polyG and polyC repeats. However, surjection to hg38 coordinates split a WT1 driver indel call in a low-complexity region into two separate calls. Validation showed that all somatic structural variant calls, except NUP98-NSD1 and FLT3-ITD, were germline variants or artifacts. Conclusions: The use of individual-specific pangenome led to fewer false-positive indel and driver calls, mainly in repeats and single cells, and changed the branching of the inferred phylogeny. However, surjection artifacts and false-positive somatic structural variants show that pangenome-based results need read-level inspection and validation before clinical use.

genomics↗

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

Panomap: Unbiased Nanopore Signal Mapping with Pangenome Variation Graphs

MotivationSignal-space nanopore mappers enable real-time mapping and filtering decisions directly from raw nanopore signals. However, existing signal-space mappers are built around linear references, and using a single representative reference can introduce reference bias when the sample diverges from that reference. Pangenome reference collections can reduce this bias by representing diversity across related reference sequences, but linear-reference signal mappers must treat each sequence as a separate target, redundantly storing shared sequences. Pangenome variation graphs provide a more compact representation by storing shared sequences once and encoding variants as alternative paths through the graph. Although sequence-to-graph mapping is well established for basecalled reads, existing signal-space methods do not directly use pangenome variation graphs. ResultsWe present Panomap, the first signal-space mapper that operates on pangenome variation graphs. Panomap maps raw nanopore signals to graph references, allowing signal-space mapping to use pangenome diversity while representing shared sequences once. We evaluate Panomap in three settings. First, when a single reference already maps the sample well, Panomap preserves mapping accuracy as additional reference sequences are added to the reference collection, while state-of-the-art signal-space tools regress. Second, when the exact sample strain is absent from the reference collection, Panomap benefits from adding related assemblies from the same species to the pangenome reference. Third, using a highly polymorphic locus, we show that Panomap can map reads from alleles not represented in the reference collection by using related alleles in the pangenome, with the largest gains for more divergent alleles and for decisions made from short prefixes of the read signal. In addition, Panomaps graph index scales sublinearly with pangenome collection size. Together, these results show that Panomap brings population-aware reference representation into signal-space mapping. Availability and ImplementationPanomap is open source and available at https://github.com/cornell-brg/panomap. Contactps2229@cornell.edu

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

A Pan-pangenome illuminates complex structural variation and selection in humans, chimpanzees, and bonobos

Complete, haplotype-resolved genome assemblies have provided unprecedented insight into the evolution of structurally complex, rapidly evolving regions of human genomes1-6; however, population-scale pangenome resources of our closest relatives, chimpanzees and bonobos (genus, Pan), are necessary to ascertain the origins and evolutionary context of these loci. Here, we sequence and assemble 58 haplotypes from four distinct Pan clades to high contiguity (median contig NG50=54 Mb), including eight near-T2T genomes. These genomes reveal previously intractable genetic variation increasing estimates of genome-wide genetic diversity 6-37% across populations compared to short-read estimates7. We identify recurrent structural polymorphisms across species impacting genes associated with immune response and host-pathogen interaction and find that structural variants (SVs) are 170- to 260-fold more likely than single nucleotide variants (SNVs) to exhibit high-impact effects across species. Contrasting SV patterns across primates we find that transposable element mutation rates differ by as much as threefold between species. We show that human disease-associated short tandem repeat (TR) loci have uniquely expanded in humans sensitizing our species to these TR-expansion disorders. Physically phased haplotypes enable reconstruction of genome-wide genealogical histories, uncovering ancient, functional genetic variation maintained by balancing selection, as well as signatures of recent adaptation in chimpanzee subspecies. Several malaria-associated loci exhibit ancient structural polymorphism, including the African great ape-specific glycophorin (GYP) gene expansion. We characterize the sequence, structure, and composition of diverse glycophorin haplotypes in humans and chimpanzees. We identify independent malaria-protective GYPA-B fusion events in humans and novel chimpanzee glycophorin genes resulting from both ancient and recent fusion events demonstrating parallel adaptations to pathogen resistance across hominins. Together, our resource highlights the critical importance of nonhuman primate population-scale pangenomics for understanding the evolution of complex genome structures and the biodiversity of our endangered closest living relatives.

evolutionary biology↗

bioRxiv2026

Population-scale Y chromosome assemblies reveal recurrent remodeling within constrained architectures

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.

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

Adaptive Tracepoints for Pangenome Alignment Compression

MotivationStoring millions of sequence alignments from large-scale genomic comparisons requires efficient compression methods. While fixed-size alignment encodings offer uniform spacing and bounded reconstruction cost, they cannot adapt to variable alignment complexity across sequences, missing compression opportunities in conserved regions. ResultsWe present adaptive tracepoints, a complexity-aware alignment encoding that segments alignments using configurable complexity metrics (edit distance or diagonal distance) rather than fixed intervals. Segments are bounded by either the number of differences or the deviation from the main diagonal, adapting to local alignment characteristics. Reconstruction guarantees that alignments maintain identical or improved alignment scores. We validate the correctness of our method on simulated and real pangenomes with varying lengths and divergences. Diagonal-bounded tracepoints achieve 10.5-13.7x better compression than fixed-length encodings (l=100) on simulated long sequence alignments (100 Kb), while edit-bounded tracepoints provide a tunable trade-off between compression and reconstruction cost, approaching diagonal-bounded compression at higher thresholds with substantially lower memory and runtime. On real pangenomes (390M alignments), these methods compress alignments by 23-139x relative to uncompressed representations, with no score degradation and reconstruction time linear in alignment length. AvailabilityCode and documentation are publicly available at https://github.com/AndreaGuarracino/tracepoints, https://github.com/AndreaGuarracino/tpa, and https://github.com/AndreaGuarracino/cigzip. Contactaguarracino@tgen.org Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2026

Population-scalable genotyping from low-coverage sequencing data using pangenome graphs

Pangenome-based genotyping of structurally complex loci remains challenging at low sequencing depths, particularly when samples are of low quality, such as in ancient DNA. We present COSIGT (COsine SImilarity-based GenoTyper), a method that infers structural genotypes by matching short-read coverage patterns to pangenome haplotypes using cosine similarity. COSIGT maintains robust accuracy at low coverage (1-2X), outperforming existing methods where depth-sensitive approaches degrade. We demonstrate scalability to thousands of modern and ancient genomes, enabling population-scale analyses of complex variation from low-coverage data.

bioinformatics↗

bioRxiv2025

Origin and evolution of acrocentric chromosomes in human and great apes

The short arms of human acrocentric chromosomes are characterized by nucleolar organizer regions essential for ribosome biogenesis, but their highly repetitive nature has hindered genomic analysis. Leveraging the recently completed genomes of all major ape lineages, we identified recurrent features of their acrocentrics, including enriched repeat classes, centromere repositioning by whole-arm inversion, interchromosomal sequence exchange, and birth-and-death evolution of multiple gene families. Together, these processes have enabled the repeated amplification and diversification of the FRG1 gene family over 25 million years of ape evolution, and, in gorilla, the formation and amplification of a novel IGSF3-GGT fusion gene under positive selection. Similar evolutionary events also explain the distribution of segmental duplications and heterochromatin in the modern human genome, predisposing it to karyotypic abnormalities such as Robertsonian translocations. Our findings highlight acrocentric chromosomes as key drivers of evolution in the great apes, with implications for speciation, adaptation, and clinical genomics.

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

The reference-free pangenome of Arabidopsis thaliana

For several decades, scientists relied on unique reference genomes as the basis for various genomic studies. This methodology presents multiple drawbacks, particularly for variation studies, often yielding unrepresentative and incomplete observations regarding the diversity of the species or group under study. Pangenomes are collections of genomic sequences from several individuals of a species or population, which allow to overcome the limitations of studies based on a single reference genome. In this study, we produced a 93-assembly pangenome of the model plant Arabidopsis thaliana using the reference-free method PanGenome Graph Builder (PGGB). The aim was to investigate the diversity within this species using this novel methodology, encompassing genomic sequences, genes, and pseudogenes. The pangenome exhibited a total length of 488.78 Mb, consisting of 30,391,243 nodes and 7,189,634 edges. We mapped a total of 2,325,577 genes and 23,894 pseudogenes across 93 assemblies, of which 36% and 0.9%, respectively, were classified as core. The observed variation in terms of genes and especially pseudogenes related to the geographical distance between sampling sites. Since the simulated pangenome growth curve based on gene did not reach a plateau, new accessions could potentially expand the represented diversity. This study enriches our understanding of intra-specific variation in A. thaliana and provides a new viewpoint on the potential applications of pangenomes in diversity research.

genomics↗

bioRxiv2025

The mouse pangenome reveals the structural complexity of the murine protein coding landscape

We present the first mouse pangenome consisting of 17 high-quality inbred mouse strain genomes with complete annotation. This collection includes 12 widely used classical laboratory strains and 5 wild-derived strains. We have fully resolved previously incomplete genomic regions, including the major histocompatibility complex (MHC), the defensin cluster, T-cell receptor, and Ly49 complexes. Hundreds of non-reference genes identified in previous publications not found in GRCm39, like Defa1, Raet1a, and Klra20 (Ly49T), were localised in the new reference genomes. We conducted the first genome-wide scan of variable number tandem repeats (VNTRs) within the coding regions of mice, identifying over 400 genes with VNTR polymorphisms up to more than 600 repeat copies and repeat units reaching 990 nucleotides. Our strain-specific annotations enhance RNA-Seq analyses, as demonstrated in PWK/PhJ, where we observed a 5.1% improvement in read mapping and expression level differences in 2.1% of coding genes compared to using GRCm39.

genomics↗

bioRxiv2025

Comparative population pangenomes reveal unexpected complexity and fitness effects of structural variants

Structural variants (SVs) are widespread in vertebrate genomes, yet their evolutionary dynamics remain poorly understood. Using 45 long-read de novo genome assemblies and pangenome tools, we analyze SVs within three closely related species of North American jays (Aphelocoma, scrub-jays) displaying a 60-fold range in effective population size. We find rapid evolution of genome architecture, including [~]100 Mb variation in genome size driven by dynamic satellite landscapes with unexpectedly long (> 10 kb) repeat units and widespread variation in gene content, influencing gene expression. SVs exhibit slightly deleterious dynamics modulated by variant length and population size, with strong evidence of adaptive fixation only in large populations. Our results demonstrate how population size shapes the distribution of SVs and the importance of pangenomes to characterizing genomic diversity.

evolutionary biology↗

bioRxiv2024

Creating a biomedical knowledge base by addressing GPT inaccurate responses and benchmarking context

We created GNQA, a generative pre-trained transformer (GPT) knowledge base driven by a performant retrieval augmented generation (RAG) with a focus on aging, dementia, Alzheimers and diabetes. We uploaded a corpus of three thousand peer reviewed publications on these topics into the RAG. To address concerns about inaccurate responses and GPT hallucinations, we implemented a context provenance tracking mechanism that enables researchers to validate responses against the original material and to get references to the original papers. To assess the effectiveness of contextual information we collected evaluations and feedback from both domain expert users and citizen scientists on the relevance of GPT responses. A key innovation of our study is automated evaluation by way of a RAG assessment system (RAGAS). RAGAS combines human expert assessment with AI-driven evaluation to measure the effectiveness of RAG systems. When evaluating the responses to their questions, human respondents give a "thumbs-up" 76% of the time. Meanwhile, RAGAS scores 90% on answer relevance on questions posed by experts. And when GPT-generates questions, RAGAS scores 74% on answer relevance. With RAGAS we created a benchmark that can be used to continuously assess the performance of our knowledge base. Full GNQA functionality is embedded in the free GeneNetwork.org web service, an open-source system containing over 25 years of experimental data on model organisms and human. The code developed for this study is published under a free and open-source software license at https://git.genenetwork.org/gn-ai/tree/README.md.

scientific communication and education↗

bioRxiv2024

The Platinum Pedigree: A long-read benchmark for genetic variants

Recent advances in genome sequencing have improved variant calling in complex regions of the human genome. However, it is difficult to quantify variant calling performance since existing standards often focus on specificity, neglecting completeness in difficult to analyze regions. To create a more comprehensive truth set, we used Mendelian inheritance in a large pedigree (CEPH-1463) to filter variants across Illumina, PacBio high-fidelity (HiFi), and Oxford Nanopore Technologies platforms. This generated a variant map with over 4.7 million single-nucleotide variants, 767,795 indels, 537,486 tandem repeats, and 24,315 structural variants, covering 2.77 Gb of the GRCh38 genome. This work adds [~]200 Mb of high-confidence regions, including 8% more small variants, and introduces the first tandem repeat and structural variant truth sets for NA12878. As an example of the value of this improved benchmark, we retrained DeepVariant using this data to reduce genotyping errors by [~]34%.

genomics↗

bioRxiv2024

The formation and propagation of human Robertsonian chromosomes

Robertsonian chromosomes are a type of variant chromosome found commonly in nature. Present in one in 800 humans, these chromosomes can underlie infertility, trisomies, and increased cancer incidence. Recognized cytogenetically for more than a century, their origins have remained mysterious. Recent advances in genomics allowed us to assemble three human Robertsonian chromosomes completely. We identify a common breakpoint and epigenetic changes in centromeres that provide insight into the formation and propagation of common Robertsonian translocations. Further investigation of the assembled genomes of chimpanzee and bonobo highlights the structural features of the human genome that uniquely enable the specific crossover event that creates these chromosomes. Resolving the structure and epigenetic features of human Robertsonian chromosomes at a molecular level paves the way to understanding how chromosomal structural variation occurs more generally, and how chromosomes evolve.

genomics↗