Biology subjects

Audano, P. A.

Publications and source records attributed to Audano, P. A..

18 recordsLinked to original sources

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↗

bioRxiv2025

Segmental duplication-mediated rearrangements alter the landscape of mouse genomes

Segmental duplications (SDs) are dynamic regions of mammalian genomes that drive structural variation and phenotypic diversity. However, genome-wide characterization of the SD landscape and the rearrangements mediated by SD paralogs in mouse genomes has been limited by the near-exclusive reliance on short-read sequencing technologies. Here, we integrate long-read genome assemblies, optical mapping, and k-mer based copy number analysis across eight genetically diverse inbred mouse strains to identify and characterize SD-mediated rearrangements. We identify 223 rearrangements affecting over 14 Mb of sequence and reveal substantial variation in gene content. These rearrangements affect loci involved in immunity, sensory perception, and gene regulation, including variation in the amylase gene cluster (Amy2a) and KRAB-zinc finger genes. We observed that SD flanks in mouse genomes are significantly enriched for young LINE-1 transposable elements, suggesting a potential role for transposons in promoting recombination and generating SDs within mice. Our findings highlight the contribution of SDs to genome structure and intra-species variation, and provide a resource for identifying regions prone to rearrangement in a critical model organism for biomedical research.

genomics↗

bioRxiv2024

Large complex structural rearrangements in human genomes harbor cryptic structures

Structural variation is a major contributor to human diversity, adaptation, and disease. Simple structural variant (SV) types include deletions, insertions, duplications, inversions, and translocations, and SVs account for most of the variable bases between genomes. Complex structural variants (CSVs) that consist of one or more simple events in cis appear more frequently in diseases and cancers where DNA repair, apoptosis, and cell cycle checkpoints are compromised, although CSVs can also appear in germline genome sequences of healthy individuals. CSVs are often characterized by short tracts of homology or no homology, and while CSVs are more prevalent in complex regions that contain large repeats, smaller stretches of homology can also enable their formation across more unique loci. Long-read assemblies have increased the size of detectable SVs and expanded variant detection into more complex regions of the genome, and while they reconstruct CSVs, methods for identifying CSVs from assemblies is limited. Here, we have developed a new assembly-based approach to trace through complex loci rather than relying upon reference representations of alignments. We can now access CSVs in large complex segmental duplications, reveal structures that were previously unknown, and identify SV breakpoints with greater accuracy. We find 72 large CSVs per genome and 128 unique complex structures and CSVs in highly repetitive regions can now be detected including several distinct complex events in repetitive NBPF genes that was not previously callable with short-read or long-read CSV methods. This approach is implemented within a key assembly-based variant calling tool, PAV, and represents a substantial improvement identifying complex variants now ascertainable from contiguous genome assemblies.

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 de novo variant in PAK2 detected in an individual with Knobloch type 2 syndrome

P21-activated kinase 2 (PAK2) is a serine/threonine kinase essential for a variety of cellular processes including signal transduction, cellular survival, proliferation, and migration. A recent report proposed monoallelic PAK2 variants cause Knobloch syndrome type 2 (KNO2)--a developmental disorder primarily characterized by ocular anomalies. Here, we identified a novel de novo heterozygous missense variant in PAK2, NM_002577.4:c.1273G>A, p.(D425N), by whole genome sequencing in an individual with features consistent with KNO2. Notable clinical phenotypes include global developmental delay, congenital retinal detachment, mild cerebral ventriculomegaly, hypotonia, FTT, pyloric stenosis, feeding intolerance, patent ductus arteriosus, and mild facial dysmorphism. The p.(D425N) variant lies within the protein kinase domain and is predicted to be functionally damaging by in silico analysis. Previous clinical genetic testing did not report this variant due to unknown relevance of PAK2 variants at the time of testing, highlighting the importance of reanalysis. Our findings also substantiate the candidacy of PAK2 variants in KNO2 and expand the KNO2 clinical spectrum.

genomics↗

bioRxiv2023

Small allelic variants are a source of ancestral bias in structural variant breakpoint placement

High-quality genome assemblies and sophisticated algorithms have increased sensitivity for a wide range of variant types, and breakpoint accuracy for structural variants (SVs, [≥] 50 bp) has improved to near basepair precision. Despite these advances, many SVs in unique regions of the genome are subject to systematic bias that affects breakpoint location. This ambiguity leads to less accurate variant comparisons across samples, and it obscures true breakpoint features needed for mechanistic inferences. To understand why SVs are not consistently placed, we re-analyzed 64 phased haplotypes constructed from long-read assemblies released by the Human Genome Structural Variation Consortium (HGSVC). We identified variable breakpoints for 882 SV insertions and 180 SV deletions not anchored in tandem repeats (TRs) or segmental duplications (SDs). While this is unexpectedly high for genome assemblies in unique loci, we find read-based callsets from the same sequencing data yielded 1,566 insertions and 986 deletions with inconsistent breakpoints also not anchored in TRs or SDs. When we investigated causes for breakpoint inaccuracy, we found sequence and assembly errors had minimal impact, but we observed a strong effect of ancestry. We confirmed that polymorphic mismatches and small indels are enriched at shifted breakpoints and that these polymorphisms are generally lost when breakpoints shift. Long tracts of homology, such as SVs mediated by transposable elements, increase the likelihood of imprecise SV calls and the distance they are shifted. Tandem Duplication (TD) breakpoints are the most heavily affected SV class with 14% of TDs placed at different locations across haplotypes. While graph genome methods normalize SV calls across many samples, the resulting breakpoints are sometimes incorrect, highlighting a need to tune graph methods for breakpoint accuracy. The breakpoint inconsistencies we characterize collectively affect [~]5% of the SVs called in a human genome and underscore a need for algorithm development to improve SV databases, mitigate the impact of ancestry on breakpoint placement, and increase the value of callsets for investigating mutational processes.

bioinformatics↗

bioRxiv2023

Whole-genome long-read sequencing downsampling and its effect on variant calling precision and recall

Advances in long-read sequencing (LRS) technology continue to make whole-genome sequencing more complete, affordable, and accurate. LRS provides significant advantages over short-read sequencing approaches, including phased de novo genome assembly, access to previously excluded genomic regions, and discovery of more complex structural variants (SVs) associated with disease. Limitations remain with respect to cost, scalability, and platform-dependent read accuracy and the tradeoffs between sequence coverage and sensitivity of variant discovery are important experimental considerations for the application of LRS. We compare the genetic variant calling precision and recall of Oxford Nanopore Technologies (ONT) and PacBio HiFi platforms over a range of sequence coverages. For read-based applications, LRS sensitivity begins to plateau around 12-fold coverage with a majority of variants called with reasonable accuracy (F1 score above 0.5), and both platforms perform well for SV detection. Genome assembly increases variant calling precision and recall of SVs and indels in HiFi datasets with HiFi outperforming ONT in quality as measured by the F1 score of assembly-based variant callsets. While both technologies continue to evolve, our work offers guidance to design cost-effective experimental strategies that do not compromise on discovering novel biology.

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↗

bioRxiv2023

Utility of long-read sequencing for All of Us

The All of Us (AoU) initiative aims to sequence the genomes of over one million Americans from diverse ethnic backgrounds to improve personalized medical care. In a recent technical pilot, we compared the performance of traditional short-read sequencing with long-read sequencing in a small cohort of samples from the HapMap project and two AoU control samples representing eight datasets. Our analysis revealed substantial differences in the ability of these technologies to accurately sequence complex medically relevant genes, particularly in terms of gene coverage and pathogenic variant identification. We also considered the advantages and challenges of using low coverage sequencing to increase sample numbers in large cohort analysis. Our results show that HiFi reads produced the most accurate results for both small and large variants. Further, we present a cloud-based pipeline to optimize SNV, indel and SV calling at scale for long-reads analysis. These results will lead to widespread improvements across AoU.

bioinformatics↗

bioRxiv2022

Assembly of 43 diverse human Y chromosomes reveals extensive complexity and variation

The prevalence of highly repetitive sequences within the human Y chromosome has led to its incomplete assembly and systematic omission from genomic analyses. Here, we present long-read de novo assemblies of 43 diverse Y chromosomes spanning 180,000 years of human evolution, including two from deep-rooted African Y lineages, and report remarkable complexity and diversity in chromosome size and structure, in contrast with its low level of base substitution variation. The size of the Y chromosome assemblies varies extensively from 45.2 to 84.9 Mbp and include, on average, 81 kbp of novel sequence per Y chromosome. Half of the male-specific euchromatic region is subject to large inversions with a >2-fold higher recurrence rate compared to inversions in the rest of the human genome. Ampliconic sequences associated with these inversions further show differing mutation rates that are sequence context-dependent and some ampliconic genes show evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, the Yq12, is composed of alternating arrays of DYZ1 and DYZ2 repeat units that show extensive variation in the number, size and distribution of these arrays, but retain a 1:1 copy number ratio of the monomer repeats, consistent with the notion that functional or evolutionary forces are acting on this chromosomal region. Finally, our data suggests that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kbp distal to the currently established boundary. The availability of sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of specific traits with Y-chromosomal variants and garnering novel insights into the evolution and function of complex regions of the human genome.

genomics↗

bioRxiv2022

Resolution of structural variation in diverse mouse genomes reveals chromatin remodeling due to transposable elements

Diverse inbred mouse strains are among the foremost models for biomedical research, yet genome characterization of many strains has been fundamentally lacking in comparison to human genomics research. In particular, the discovery and cataloging of structural variants is incomplete, limiting the discovery of potentially causative alleles for phenotypic variation across individuals. Here, we utilized long-read sequencing to resolve genome-wide structural variants (SVs, variants [≥] 50 bp) in 20 genetically distinct inbred mice. We report 413,758 site-specific SVs that affect 13% (356 Mbp) of the current mouse reference assembly, including 510 previously unannotated variants which alter coding sequences. We find that 39% of SVs are attributed to transposable element (TE) variation accounting for 75% of bases altered by SV. We then utilized this callset to investigate the impact of TE heterogeneity on mouse embryonic stem cells (mESCs), and find multiple TE classes that influence chromatin accessibility across loci. We also identify strain-specific transcription start sites originating in polymorphic TEs that modify gene expression. Our work provides the first long-read based analysis of mouse SVs and illustrates that previously unresolved TEs underlie epigenetic and transcriptome differences in mESCs.

genomics↗

bioRxiv2022

Transposable element-mediated rearrangements are prevalent in human genomes

Transposable elements constitute about half of human genomes, and their role in generating human variation through retrotransposition is broadly studied and appreciated. Structural variants mediated by transposons, which we call transposable element-mediated rearrangements (TEMRs), are less well studied, and the mechanisms leading to their formation as well as their broader impact on human diversity are poorly understood. Here, we identify 493 unique TEMRs across the genomes of three individuals. While homology directed repair is the dominant driver of TEMRs, our sequence-resolved TEMR resource allows us to identify complex inversion breakpoints, triplications or other high copy number polymorphisms, and additional complexities. TEMRs are enriched in genic loci and can create potentially important risk alleles such as a deletion in TRIM65, a known cancer biomarker and therapeutic target. These findings expand our understanding of this important class of structural variation, the mechanisms responsible for their formation, and establish them as an important driver of human diversity.

genomics↗

bioRxiv2021

Haplotype-resolved inversion landscape reveals hotspots of mutational recurrence associated with genomic disorders

Unlike copy number variants (CNVs), inversions remain an underexplored genetic variation class. By integrating multiple genomic technologies, we discover 729 inversions in 41 human genomes. Approximately 85% of inversions <2 kbp form by twin-priming during L1-retrotransposition; 80% of the larger inversions are balanced and affect twice as many base pairs as CNVs. Balanced inversions show an excess of common variants, and 72% are flanked by segmental duplications (SDs) or mobile elements. Since this suggests recurrence due to non-allelic homologous recombination, we developed complementary approaches to identify recurrent inversion formation. We describe 40 recurrent inversions encompassing 0.6% of the genome, showing inversion rates up to 2.7x10-4 per locus and generation. Recurrent inversions exhibit a sex- chromosomal bias, and significantly co-localize to the critical regions of genomic disorders. We propose that inversion recurrence results in an elevated number of heterozygous carriers and structural SD diversity, which increases mutability in the population and predisposes to disease- causing CNVs.

genomics↗

bioRxiv2020

De novo assembly of 64 haplotype-resolved human genomes of diverse ancestry and integrated analysis of structural variation

Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent-child trio data. We present 64 assembled haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average contig N50: 26 Mbp) integrate all forms of genetic variation across even complex loci such as the major histocompatibility complex. We focus on 107,590 structural variants (SVs), of which 68% are inaccessible by short-read sequencing. We identify new SV hotspots (spanning megabases of gene-rich sequence), characterize 130 of the most active mobile element source elements, and find that 63% of all SVs arise by homology-mediated mechanisms--a twofold increase from previous studies. Our resource now enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1,525 expression quantitative trait loci (SV-eQTLs) as well as SV candidates for adaptive selection within the human population.

genetics↗

bioRxiv2020

Pangenome-based genome inference

AO_SCPLOWBSTRACTC_SCPLOWTypical analysis workflows map reads to a reference genome in order to detect genetic variants. Generating such alignments introduces references biases, in particular against insertion alleles absent in the reference and comes with substantial computational burden. In contrast, recent k-mer-based genotyping methods are fast, but struggle in repetitive or duplicated regions of the genome. We propose a novel algorithm, called PanGenie, that leverages a pangenome reference built from haplotype-resolved genome assemblies in conjunction with k-mer count information from raw, short-read sequencing data to genotype a wide spectrum of genetic variation. The given haplotypes enable our method to take advantage of linkage information to aid genotyping in regions poorly covered by unique k-mers and provides access to regions otherwise inaccessible by short reads. Compared to classic mapping-based approaches, our approach is more than 4x faster at 30x coverage and at the same time, reached significantly better genotype concordances for almost all variant types and coverages tested. Improvements are especially pronounced for large insertions (> 50bp), where we are able to genotype > 99.9% of all tested variants with over 90% accuracy at 30x short-read coverage, where the best competing tools either typed less than 60% of variants or reached accuracies below 70%. PanGenie now enables the inclusion of this commonly neglected variant type in downstream analyses.

bioinformatics↗

bioRxiv2020

Expectations and blind spots for structural variation detection from short-read alignment and long-read assembly

Virtually all genome sequencing efforts in national biobanks, complex and Mendelian disease programs, and emerging clinical diagnostic approaches utilize short-reads (srWGS), which present constraints for genome-wide discovery of structural variants (SVs). Alternative long-read single molecule technologies (lrWGS) offer significant advantages for genome assembly and SV detection, while these technologies are currently cost prohibitive for large-scale disease studies and clinical diagnostics (∼5-12X higher cost than comparable coverage srWGS). Moreover, only dozens of such genomes are currently publicly accessible by comparison to millions of srWGS genomes that have been commissioned for international initiatives. Given this ubiquitous reliance on srWGS in human genetics and genomics, we sought to characterize and quantify the properties of SVs accessible to both srWGS and lrWGS to establish benchmarks and expectations in ongoing medical and population genetic studies, and to project the added value of SVs uniquely accessible to each technology. In analyses of three trios with matched srWGS and lrWGS from the Human Genome Structural Variation Consortium (HGSVC), srWGS captured ∼11,000 SVs per genome using reference-based algorithms, while haplotype-resolved assembly from lrWGS identified ∼25,000 SVs per genome. Detection power and precision for SV discovery varied dramatically by genomic context and variant class: 9.7% of the current GRCh38 reference is defined by segmental duplications (SD) and simple repeats (SR), yet 91.4% of deletions that were specifically discovered by lrWGS localized to these regions. Across the remaining 90.3% of the human reference, we observed extremely high concordance (93.8%) for deletions discovered by srWGS and lrWGS after error correction using the raw lrWGS reads. Conversely, lrWGS was superior for detection of insertions across all genomic contexts. Given that the non-SD/SR sequences span 90.3% of the GRCh38 reference, and encompass 95.9% of coding exons in currently annotated disease associated genes, improved sensitivity from lrWGS to discover novel and interpretable pathogenic deletions not already accessible to srWGS is likely to be incremental. However, these analyses highlight the added value of assembly-based lrWGS to create new catalogues of functional insertions and transposable elements, as well as disease associated repeat expansions in genomic regions previously recalcitrant to routine assessment.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗

bioRxiv2019

A fully phased accurate assembly of an individual human genome

The prevailing genome assembly paradigm is to produce consensus sequences that "collapse" parental haplotypes into a consensus sequence. Here, we leverage the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing (Strand-seq)1,2 and combine them with high-fidelity (HiFi) long sequencing reads3, in a novel reference-free workflow for diploid de novo genome assembly. Employing this strategy, we produce completely phased de novo genome assemblies separately for each haplotype of a single individual of Puerto Rican origin (HG00733) in the absence of parental data. The assemblies are accurate (QV > 40), highly contiguous (contig N50 > 25 Mbp) with low switch error rates (0.4%) providing fully phased single-nucleotide variants (SNVs), indels, and structural variants (SVs). A comparison of Oxford Nanopore and PacBio phased assemblies identifies 150 regions that are preferential sites of contig breaks irrespective of sequencing technology or phasing algorithms.

bioinformatics↗

bioRxiv2019

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

The sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective stand-alone technology for de novo assembly of human genomes.

genomics↗