Biology subjects

Eberle, M. A.

Publications and source records attributed to Eberle, M. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Resolution of the D4Z4 repeat responsible for facioscapulohumeral muscular dystrophy with HiFi sequencing

The D4Z4 macrosatellite repeat encompasses some of the most difficult-to-resolve disease-related variations in the human genome. D4Z4 has a repeat unit of 3.3 kb (encoding the DUX4 gene) that is present in up to 100 copies on two chromosomes (4 and 10), while DUX4 can only be expressed in somatic cells from the permissive A haplotype that usually occurs on chromosome 4. Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation and ectopic expression of DUX4 in skeletal muscle, mediated by contraction of D4Z4 to 1-10 copies (FSHD1, 95% of FSHD cases) or mutations in chromatin factor genes such as SMCHD1 (FSHD2, 5% of FSHD cases). Due to its large size, disease specific haplotypes and sequence homology between chromosomes, D4Z4 is challenging to resolve by current sequencing technologies. We report a computational tool, Kivvi, to genotype D4Z4 using PacBio whole-genome long-read sequence data. Kivvi detects all D4Z4 alleles in a sample, reporting the repeat size, chromosome (4 vs. 10), distal haplotype (A vs. non-permissive haplotypes) and the methylation level of each allele. We validated Kivvi against gold standard assays for FSHD diagnostics, detecting 100% of contracted alleles and correctly classifying 90% of noncontracted alleles. We showed differential methylation signals between FSHD1 and candidate FSHD2 samples. We profiled D4Z4 across 601 individuals from five ancestral populations, revealing extensive genetic diversity. We identified common haplotypes of D4Z4 alleles and characterized hybrid repeat units, hybrid repeat arrays, and translocation alleles. Combined with HiFi long reads, Kivvi enables the consolidation of multiple FSHD assays into a single workflow and facilitates the discovery of novel genetic modifiers of FSHD through population-scale studies.

genetics↗

bioRxiv2026

A computational model for quantifying instability of tandem repeats across the genome

Tandem repeats (TRs) exhibit high levels of somatic mosaicism, which is increasingly recognized as an important modifier of repeat expansion disorders. Long-read sequencing can capture full-length repeat alleles, yet robust frameworks for quantifying instability across TRs genome-wide are still needed. Here, we introduce a general-purpose model for quantifying TR instability in a given long-read sequencing dataset, without explicitly distinguishing biological mosaicism from technical noise, and which is broadly applicable to both simple and structurally complex loci. This model accurately characterizes allelic instability at each TR locus by representing the distribution of read-to-consensus deviations for each allele. Using HiFi sequencing data from 256 HPRC cell line samples, we fitted models for 617,007 TR loci, including known pathogenic repeats. We observe that instability levels are generally low, but vary substantially across individual TRs, and are driven more strongly by repeat composition than overall repeat length. Furthermore, we applied our method to targeted PureTarget long-read data from samples with known repeat expansions and identified significant mosaicism in the majority of expanded alleles. Our model offers a practical way to quantify instability of tandem repeats across the genome and to detect unusually unstable repeat alleles.

bioinformatics↗

bioRxiv2026

Hunting for microsatellite instability in long-read data with Owl

Microsatellite instability (MSI) is a key biomarker of mismatch repair deficiency and response to immunotherapy, yet most existing genomic detection methods are optimized for short-read sequencing and rely on small panels of homopolymer markers, limiting the ability to characterize genome-wide and motif-specific patterns of instability. Here we present Owl, a bioinformatic tool for quantifying MSI from long-read (PacBio) genomic data. Owl leverages a genome-wide marker set of more than 140,000 microsatellite repeats ranging from 1-6 bp in length to measure MSI across a phased genome. Using a wrap-around alignment algorithm, Owl constructs repeat-length distributions at each marker site and flags somatic instability using the coefficient of variation. We applied Owl to screen for markers with stable coverage, phasing, and baseline variation across 131 diverse genomes from the Human Pangenome Reference Consortium, where Owl scores ranged from 1.4% to 5.4% of markers exceeding the instability threshold. When applied to 19 cancer cell lines and one diffuse astrocytoma tumor-normal pair, Owl identified five MSI-high genomes with 15-18% unstable markers and showed close concordance with an Illumina DRAGEN MSI assay for the astrocytoma sample. Motif-level analyses revealed shared enrichment of short homopolymer and dinucleotide (A- and AT-rich) repeats across MSI-high cancers, and additionally uncovered a distinct pattern of elevated GGAA microsatellite instability in Ewing sarcoma cell lines, consistent with the known role of the EWS::FLI1 fusion protein at GGAA-rich regulatory elements. Owl is implemented in Rust and integrated into the PacBio HiFi Somatic workflow, providing a scalable framework for MSI analysis from long-read sequencing focused on repeat instability specifically in tumor samples.

bioinformatics↗

bioRxiv2025

Aardvark: Sifting through differences in a mound of variants

Variant benchmarking is critical in assessing the accuracy of genomic secondary pipelines. However, traditional benchmarking tools that require exact genotype matches inject biases from variant representation and are ill-suited for tandem repeat or structural variation. We describe Aardvark, a variant benchmarking tool that introduces the basepair score to directly compare haplotype sequences, reducing representation biases while allowing for partial credit scoring. The tool also includes a traditional genotype score and supports separate or joint benchmarking of small variants, tandem repeats, and structural variants (<10 kb). Aardvark accepts standard inputs, runs {approx}16x faster than hap.py, and is freely available and open source (https://github.com/PacificBiosciences/aardvark).

bioinformatics↗

bioRxiv2025

Complex structural variant visualization with SVTopo

Structural variants are genomic variants that impact at least 50 nucleotides and can play major roles in diversity and human health. Many structural variants are complex multi-breakpoint rearrangements that are difficult to comprehend with existing visualization tools. We present SVTopo, a tool to visualize complex structural variants with supporting evidence from high-accuracy long reads, in easily understood figures. We include examples of eleven categories of complex structural variants from seven human genomes. SVTopo shows breakpoint evidence in ways that aid reasoning about the impact of large, multi-breakpoint events such as inversions, translocations, and combinations of simpler structural variants.

bioinformatics↗

bioRxiv2025

Detailed tandem repeat allele profiling in 1,027 long-read genomes reveals genome-wide patterns of pathogenicity

Short tandem repeat (STR) instability is a major cause of disease association, yet their full length-distributions and sequence content have been difficult to resolve with short-read sequencing. By analyzing long-read sequencing genomes from 2,645 participants of the All of Us Research Program, we establish a comprehensive STR resource of 23 billion alleles covering a catalog of 4.4 million loci. We find that known disease-associated STR loci exhibit markedly elevated allelic variability relative to genome-wide distributions, which is most pronounced when based on the length of uninterrupted, pure repeat motifs (Odds Ratio: 244, p=1.62e-29). We establish the Pure Length Variability Index (PLVI) as a measure of this instability, which is robust across ancestries and replicated in 500 samples from the 1000 Genomes Project (Odds Ratio: 84, p=2.72e-28). To illustrate, we identify 55 unstable coding CAG and 5-UTR CGG STR loci with this metric, 60% of which have not been associated with disease. From this set, we document potential pathogenic repeat expansions for FAM193B and EP400 by integrating PLVI with single-genome length outlier analysis. These data establish a population-scale framework for STR discovery in rare and common diseases using long-read sequencing cohorts.

genomics↗

bioRxiv2024

StarPhase: Comprehensive Phase-Aware Pharmacogenomic Diplotyper for Long-Read Sequencing Data

Pharmacogenomics is central to precision medicine, informing medication safety and efficacy. Phar-macogenomic diplotyping of complex genes requires full-length DNA sequences and detection of structural rearrangements. We introduce StarPhase, a tool that leverages PacBio HiFi sequence data to diplotype 21 CPIC Level A pharmacogenes and provides detailed haplotypes and supporting visualizations for HLA-A, HLA-B, and CYP2D6. StarPhase diplotypes have high concordance with benchmarks where 99.5% are either exact matches or minor discrepancies. Manual inspection of the 0.5% mismatches indicates they were correctly called by StarPhase. With StarPhase, we update or correct 26.2% of GeT-RM pharmacogenomic diplotypes. Population distributions from StarPhase mostly reflect those of the All of Us cohort, while also highlighting gaps in existing pharmacogenomic databases that long-read sequencing can fill. With a single HiFi whole genome sequencing assay, StarPhase enables robust PGx diplotyping even as additional pharmacogenes and haplotypes are discovered.

bioinformatics↗

bioRxiv2024

Defining a tandem repeat catalog and variation clusters for genome-wide analyses and population databases

Tandem repeat (TR) catalogs are important components of repeat genotyping studies as they define the genomic coordinates and expected motifs of all TR loci being analyzed. In recent years, genome-wide studies have used catalogs ranging in size from fewer than 200,000 to over 7 million loci. Where these catalogs overlapped, they often disagreed on locus boundaries, hindering the comparison and reuse of results across studies. Now, with multiple groups developing public databases of TR variation in large population cohorts, there is a risk that, without sufficient consensus in the choice of locus definitions, the use of divergent repeat catalogs will lead to confusion, fragmentation, and incompatibility across resources. In this paper, we compare existing TR catalogs and discuss desirable features of a comprehensive genome-wide catalog. We then present a new, richly annotated catalog designed for large-scale analyses and population databases. This new catalog, which we call the TRExplorer catalog v1.0, contains 4.86 million TR loci and, unlike most catalogs, is designed to be useful for both short-read and long-read analyses. It consists of 4,803,366 STRs and 59,675 VNTRs, of which 780,607 STRs and 21,888 VNTRs are both polymorphic and entirely absent from widely-used catalogs previously developed for short-read analyses. Additionally, our catalog stratifies TRs into two groups: 1) isolated TRs suitable for repeat copy number analysis using short-read or long-read data and 2) so-called variation clusters that contain TRs within wider polymorphic regions that are best studied through sequence-level analysis. To define variation clusters, we present a novel algorithm that leverages long-read HiFi sequencing data to group repeats with surrounding polymorphisms. We show that the human genome contains at least 25,000 complex variation clusters, most of which span over 120 bp and contain five or more TRs. Resolving the sequence of entire variation clusters instead of individually genotyping constituent TRs leads to a more accurate analysis of these regions and enables us to profile variation that would have been missed otherwise. We also share the trexplorer.broadinstitute.org portal which allows anyone to search, visualize, and download the catalog along with variation clusters and annotations.

genomics↗

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

Sawfish: Improving long-read structural variant discovery and genotyping with local haplotype modeling

MotivationStructural variants (SVs) play an important role in evolutionary and functional genomics but are challenging to characterize. High-accuracy, long-read sequencing can substantially improve SV characterization when coupled with effective calling methods. While state-of the-art long-read SV callers are highly accurate, further improvements are achievable by systematically modeling local haplotypes during SV discovery and genotyping. ResultsWe describe sawfish, an SV caller for mapped high-quality long reads incorporating systematic SV haplotype modeling to improve accuracy and resolution. Assessment against the draft Genome in a Bottle (GIAB) SV benchmark from the T2T-HG002-Q100 diploid assembly shows that sawfish has the highest accuracy among state-of-the-art long-read SV callers across every tested SV size group. Additionally, sawfish maintains the highest accuracy at every tested depth level from 10 to 32-fold coverage, such that other callers required at least 30-fold coverage to match sawfish accuracy at 15-fold coverage. Sawfish also shows the highest accuracy in the GIAB challenging medically relevant genes benchmark, demonstrating improvements in both comprehensive and medically relevant contexts. When joint-genotyping 10 samples from CEPH-1463, sawfish has over 9000 more pedigree-concordant calls than other state-of-the-art SV callers, with the highest proportion of concordant SVs (78%) as well. Sawfishs quality model can be used to select for an even higher proportion of concordant SVs (86%), while still calling over 5000 more pedigree-concordant SVs than other callers. These results demonstrate that sawfish improves on the state-of-the-art for long-read SV calling accuracy across both individual and joint-sample analyses. AvailabilitySawfish is released as a pre-compiled Linux binary and user guide on GitHub: https://github.com/PacificBiosciences/sawfish.

bioinformatics↗

bioRxiv2024

TRGT-denovo: accurate detection of de novo tandem repeat mutations

MotivationIdentifying de novo tandem repeat (TR) mutations on a genome-wide scale is essential for understanding genetic variability and its implications in rare diseases. While PacBio HiFi sequencing data enhances the accessibility of the genomes TR regions for genotyping, simple de novo calling strategies often generate an excess of likely false positives, which can obscure true positive findings, particularly as the number of surveyed genomic regions increases. ResultsWe developed TRGT-denovo, a computational method designed to accurately identify all types of de novo TR mutations--including expansions, contractions, and compositional changes-- within family trios. TRGT-denovo directly interrogates read evidence, allowing for the detection of subtle variations often overlooked in variant call format (VCF) files. TRGT-denovo improves the precision and specificity of de novo mutation (DNM) identification, reducing the number of de novo candidates by an order of magnitude compared to genotype-based approaches. In our experiments involving eight rare disease trios previously studied TRGT-denovo correctly reclassified all false positive DNM candidates as true negatives. Using an expanded repeat catalog, it identified new candidates, of which 95% (19/20) were experimentally validated, demonstrating its effectiveness in minimizing likely false positives while maintaining high sensitivity for true discoveries. Availability and implementationBuilt in Rust, TRGT-denovo is available as source code and a pre-compiled Linux binary along with a user guide at: https://github.com/PacificBiosciences/trgt-denovo.

bioinformatics↗

bioRxiv2024

Genome-wide profiling of highly similar paralogous genes using HiFi sequencing

Variant calling is hindered in segmental duplications by sequence homology. We developed Paraphase, a HiFi-based informatics method that resolves highly similar genes by phasing all haplotypes of a gene family. We applied Paraphase to 160 long (>10 kb) segmental duplication regions across the human genome with high (>99%) sequence similarity, encoding 316 genes. Analysis across five ancestral populations revealed highly variable copy numbers of these regions. We identified 23 families with exceptionally low within-family diversity, where extensive gene conversion and unequal-crossing over have resulted in highly similar gene copies. Furthermore, our analysis of 36 trios identified 7 de novo SNVs and 4 de novo gene conversion events, 2 of which are non-allelic. Finally, we summarized extensive genetic diversity in 9 medically relevant genes previously considered challenging to genotype. Paraphase provides a framework for resolving gene paralogs, enabling accurate testing in medically relevant genes and population-wide studies of previously inaccessible genes.

genomics↗

bioRxiv2023

A common flanking variant is associated with enhanced meiotic stability of the FGF14-SCA27B locus

The factors driving initiation of pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14-SCA27B (GAA)*(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a 5-flanking 17-bp deletion-insertion in 70.34% of alleles (3,463/4,923). This common sequence variation was present nearly exclusively on alleles with fewer than 30 GAA-pure repeats and was associated with enhanced meiotic stability of the repeat locus.

genetics↗

bioRxiv2023

Resolving the unsolved: Comprehensive assessment of tandem repeats at scale

Tandem repeat (TR) variation is associated with gene expression changes and over 50 rare monogenic diseases. Recent advances in sequencing have enabled accurate, long reads that can characterize the full-length sequence and methylation profile of TRs. However, despite these advances in sequencing technology, computational methods to fully profile tandem repeats across the genome do not exist. To address this gap, we introduce tools for tandem repeat genotyping (TRGT), visualization and an accompanying TR database. TRGT accurately resolves the length and sequence composition of TR regions in the human genome. Assessing 937,122 TRs, TRGT showed a Mendelian concordance of 99.56%, allowing a single repeat unit difference. In six samples with known repeat expansions, TRGT detected all repeat expansions while also identifying methylation signals, mosaicism, and providing finer resolution of repeat length. Additionally, we release a database with allele sequences and methylation levels for 937,122 TRs across 100 genomes.

genomics↗

bioRxiv2023

Identification of allele-specific KIV-2 repeats and impact on Lp(a) measurements for cardiovascular disease risk

The abundance of Lp(a) protein holds significant implications for the risk of cardiovascular disease (CVD), which is directly impacted by the copy number (CN) of KIV-2, a 5.5 kbp sub-region. KIV-2 is highly polymorphic in the population and accurate analysis is challenging. In this study, we present the DRAGEN KIV-2 CN caller, which utilizes short reads. Data across 166 WGS show that the caller has high accuracy, compared to optical mapping and can further phase [~]50% of the samples. We compared KIV-2 CN numbers to 24 previously postulated KIV-2 relevant SNVs, revealing that many are ineffective predictors of KIV-2 copy number. Population studies, including USA-based cohorts, showed distinct KIV-2 CN, distributions for European-, African-, and Hispanic-American populations and further underscored the limitations of SNV predictors. We demonstrate that the CN estimates correlate significantly with the available Lp(a) protein levels and that phasing is highly important.

genomics↗

bioRxiv2022

Comprehensive SMN1 and SMN2 profiling for spinal muscular atrophy analysis using long-read PacBio HiFi sequencing

Spinal muscular atrophy, a leading cause of early infant death, is caused by biallelic mutations of the SMN1 gene. Sequence analysis of SMN1 is challenging due to high sequence similarity with its paralog SMN2. Both genes have variable copy numbers across populations. Furthermore, without pedigree information, it is impossible to identify silent carriers (2+0) with two copies of SMN1 on one chromosome and zero copies on the other. We developed Paraphase, an informatics method that identifies full-length SMN1 and SMN2 haplotypes, determines the gene copy numbers and calls phased variants using long-read PacBio HiFi data. The SMN1 and SMN2 copy number calls by Paraphase are highly concordant with orthogonal methods (99.2% for SMN1 and 100% for SMN2). We applied Paraphase to 438 samples across five ethnic populations to conduct a population-wide haplotype analysis of these highly homologous genes. We identified major SMN1 and SMN2 haplogroups and characterized their co-segregation through pedigree-based analyses. We identified two SMN1 haplotypes that form a common two-copy SMN1 allele in African populations. Testing positive for these two haplotypes in an individual with two copies of SMN1 gives a silent carrier risk of 88.5%, which is significantly higher than the currently used marker (1.7-3.0%). Extending beyond simple copy number testing, Paraphase can detect pathogenic variants and enable potential haplotype-based screening of silent carriers through statistical phasing of haplotypes into alleles. Future analysis of larger population data will allow identification of more diverse haplotypes and genetic markers for silent carriers.

genetics↗

bioRxiv2022

Huntington's disease phenocopy syndromes revisited: a clinical comparison and next-generation sequencing exploration

When the genetic test for the Huntingtons disease (HD) HTT expansion first became available almost 30 years ago, only 1% of patients tested negative. Since then, the test has become more accessible and the HD phenotype has expanded. More patients are being tested overall, and more negative tests are being received. These patients are deemed "HD phenocopy syndromes" (HDPC). In this study we established a current estimate for the prevalence of these patients. We also surveyed HD clinician experts on what would make them consider an HD test and compared both HD and HDPC patients to these expectations to decide whether they could be distinguished clinically; this proved impossible even when comparing symptom patterns. We re-analysed existing gene panel data for likely and potentially deleterious variants. Furthermore, we determined principles to prioritise patients for whole-genome sequencing (WGS). It was used to probe a 50 patient strong subcohort of HD phenocopy syndromes for known causes of HD-like and other neurodegenerative disease, identifying one ATXN1 expansion using ExpansionHunter(R). This was a small genetic substudy and therefore unsurprisingly no other known deleterious variants could be identified as in these cryptic understudied syndromes. Novel variants in known genes and variants in genes not yet linked to neurodegeneration may play an outsized role.

genetics↗

bioRxiv2022

A genome-wide atlas of recurrent repeat expansions in human cancer

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs (STRs), a phenomenon termed microsatellite instability (MSI); however larger repeat expansions have not been systematically analyzed in cancer. Here, we identified TR expansions in 2,622 cancer genomes, spanning 29 cancer types. In 7 cancer types, we found 160 recurrent repeat expansions (rREs); most of these (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with an enrichment near candidate cis-regulatory elements, suggesting a role in gene regulation. One rRE located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, targeting cells harboring this rRE with a rationally designed, sequence-specific DNA binder led to a dose-dependent decrease in cell proliferation. Overall, our results demonstrate that rREs are an important but unexplored source of genetic variation in human cancers, and we provide a comprehensive catalog for further study.

genomics↗