Biology subjects

Dashnow, H.

Publications and source records attributed to Dashnow, H..

15 recordsLinked to original sources

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

A family portrait of the genomic factors shaping tandem repeat mutagenesis

Tandem repeats (TRs) are among the most mutable loci in the human genome, but the genomic determinants of TR mutagenesis remain mysterious. We used PacBio HiFi long-read sequencing to profile nearly eight million TR loci in 28 members of a large, four-generation CEPH/Utah family designated K1463. We identified 1,270 de novo TR expansions and contractions across 20 children in the pedigree. De novo mutations (DNMs) were more likely to occur at loci that were longer, composed of uninterrupted motif sequences, and heterozygous in the parental germline. Children born to older fathers also exhibited more de novo mutations at short tandem repeats (STRs). A total of 43 TR loci were hyper-mutable in K1463, expanding or contracting up to twelve times across the pedigree. Among hyper-mutable loci that comprised multiple motifs (i.e., "complex" loci), specific motifs expanded and contracted more often than others; for example, all ten DNMs at a complex, hyper-mutable locus near the non-coding RNA LINC03021 involved the same 19bp motif. The mutability of particular motifs may be attributable to allele length, as 95% of DNMs at complex loci were expansions and contractions of the most abundant motif on a parental haplotype. However, future work will be required to disentangle the effects of nucleotide content and allele length on motif-specific mutability, especially at hyper-mutable TRs. Overall, this study combines long-read sequencing technologies with new software tools to comprehensively investigate the factors that influence TR mutagenesis.

genomics↗

bioRxiv2026

A comprehensive assessment of tandem repeat genotyping methods for Nanopore long-read genomes

BackgroundTandem repeats (TRs) play critical roles in human disease and phenotypic diversity but are among the most challenging classes of genomic variation to measure accurately. While it is possible to identify TR expansions using short-read sequencing, these methods are limited because they often cannot accurately determine repeat length or sequence composition. Long-read sequencing (LRS) has the potential to accurately characterize long TRs, including the identification of non-canonical motifs and complex structures. However, while there are an increasing number of genotyping methods available, no systematic effort has been undertaken to evaluate their length and sequence-level accuracy, performance across motifs from STRs to VNTRs and across allele lengths, and, critically, how usable these tools are in practice. ResultsWe reviewed 25 available bioinformatic tools, and selected seven that are actively maintained for benchmarking using publicly available Oxford Nanopore genome sequencing data from more than 100 individuals. Our benchmarking catalog included [~]43k TR loci genome-wide, selected to represent a range of simple and challenging TR loci. As no "truth" exists for this purpose, we used four complementary strategies to assess accuracy: concordance with high-quality haplotype-resolved Human Pangenome Reference Consortium (HPRC) assemblies, Mendelian consistency in Genome in a Bottle trios, cross-tool consistency, and sensitivity in individuals with pathogenic TR expansions confirmed by molecular methods. For all comparisons, we assess both total allele length and full sequence similarity using the Levenshtein distance. We also evaluated installation, documentation, computational requirements, and output characteristics to reflect real-world use. We provide a complete analysis workflow for all tools to support community reuse. Tool performance varied substantially across both accuracy and usability. Most methods achieved high concordance with HPRC assemblies, with higher accuracy when using the R10 ONT pore chemistry. Accuracy generally declined with increasing allele length, and most tools performed worse on homopolymers, likely reflecting underlying sequencing accuracy. Tools generally performed worse at heterozygous loci and at alleles that differed from the reference genome. Interestingly, concordance with assembly in population samples did not predict sensitivity to pathogenic expansions, with different genotypers performing best in each category. Similarly, Mendelian consistency was highest in the tool that performed worst in assembly concordance. ConclusionsNo single genotyper emerged as consistently best across all assessments, but strong contenders emerged in each. Our results demonstrate that length accuracy (a typical benchmarking approach) alone overestimates TR genotyping performance. Sequence-level benchmarking is essential for selecting tools best-suited for population studies and clinical diagnostics. This work provides practical guidance for tool selection and highlights key priorities for future long-read TR genotyping method development.

bioinformatics↗

bioRxiv2025

Long-read sequencing reveals extensive FMR1 somatic mosaicism in Fragile-X associated tremor/ataxia syndrome in human brain

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder caused by a CGG repeat expansion in the 5 untranslated region of the X-linked Fragile X messenger ribonucleoprotein 1 (FMR1). Although the CGG repeat tract is known for instability that has been posited to contribute to clinical heterogeneity, the extent of somatic variation in human brain remains unclear, in part due to the technical limitations of sequencing long tandem repeats. Here, we quantified FMR1 somatic variation in post-mortem brain tissue from individuals with FXTAS (n = 6) and Fragile X syndrome (FXS, n = 2) by applying amplification-free, targeted, long-read sequencing. This approach uncovered remarkable somatic mosaicism in repeat size and methylation in FXTAS, including somatic expansions and contractions which were not resolvable with traditional approaches. For example, in FXTAS, we identified somatic expansions to over 6000 base pairs in length as well as contractions to below the pathogenic range. We also identified unexpected patterns of methylation mosaicism on pre and full mutations. On the other hand, we replicated prior findings suggesting differential CGG expansion on the active X-chromosome in XX females. Finally, we examined the above cohort for expansions in 19 additional disease-associated repeat loci. Remarkably, we identified additional expansions in 5 out of 8 affected individuals, in FXN and RFC1. This work provides new insight into the extensive molecular variation underlying FXTAS in human brain and establishes a framework for studying repeat expansion disorders more broadly, highlighting the potential of long-read sequencing to advance our fundamental understanding of somatic mosaicism of these intractable regions of our genome.

genetics↗

bioRxiv2025

The selective dynamics of interruptions at short tandem repeats

Short tandem repeats (STRs) are hotspots of genomic instability that mutate at rates orders of magnitude greater than non-repetitive loci due to frequent replication slippage. Expansions at some STR loci cause Mendelian diseases, while variation at other noncoding loci may affect complex traits, possibly by altering transcription factor occupancy of nearby binding sites. Accordingly, some STRs are inferred to be under purifying selection, regardless of their instability. One or more "interruptions", or bases that disrupt the locuss canonical repeat, significantly decrease an STRs mutability. For example, the onset of Huntingtons Disease, a neurodegenerative disorder associated with somatic expansions of a trinucleotide coding STR, is delayed in individuals whose inherited alleles contain interruptions. Thus, interruptions that decrease mutation rate at some coding loci may broadly protect against deleterious phenotypes associated with locus instability. However, interruptions may themselves be deleterious at constrained loci, particularly at noncoding loci in gene regulatory elements, possibly disrupting the formation of secondary structures key to their function. We therefore hypothesized that the frequency of interruptions could depend on a locuss functional importance-at constrained loci, the fitness effects of expansions but also interruptions could be more deleterious than at neutral loci. To test this hypothesis, we examined the distribution of interruptions at [~]650,000 autosomal STRs. In the [~]2,500 3- or 6bp-motif coding STRs, we find that synonymous interruption density increases with purifying selection on the gene, while the opposite is true for missense-causing interruptions. In contrast, noncoding STRs in gene regulatory elements harbor fewer interruptions than those unassociated with function and putatively evolving neutrally. Our findings indicate that the abundance of interruptions may be partially explained at coding STRs by the benefit of lower instability. In contrast, maintaining a minimum core stretch of uninterrupted repeat may be key to the function of noncoding STRs that fall within regulatory elements, outweighing the benefits of stability.

evolutionary biology↗

bioRxiv2024

Polymorphic tandem repeats shape single-cell gene expression across the immune landscape

Tandem repeats (TRs) - highly polymorphic, repetitive sequences across the human genome - are important regulators of gene expression but remain underexplored due to challenges in accurate genotyping and analysis1. Here, we generate new whole genome and single-cell RNA sequencing from >5.4 million blood-derived cells across 1,925 individuals in two cohorts [Cuomo et al., accompanying manuscript], and perform meta-analysis to characterize the impact of variation in >1.7 million TR loci on immune cell type-specific gene expression. We identify >69,000 single-cell expression TR loci (sc-eTRs), 30.7% of which are specific to one of 28 immune cell types, and reveal dynamic regulatory effects using cell-state inference. Matched single-cell ATAC sequencing profiles from >3.4 million nuclei in 922 individuals [Xue et al., accompanying manuscript]. uncover chromatin accessibility QTLs for nearly one-third of expression-associated TRs, supporting coordinated effects on cis-regulatory architecture. Fine-mapping implicates 1,490 TRs as candidate causal drivers of gene expression in 6.1% of tested genes, and colocalization analyses highlight >200 genes in which TRs likely mediate genetic associations with immune and hematological traits. Together, these results provide a genome-wide, multiomic view of TR-mediated regulation in the human immune system, establishing TRs as key contributors to cell type-specific regulatory variation and complex trait architecture.

genomics↗

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

A familial, telomere-to-telomere reference for human de novo mutation and recombination from a four-generation pedigree

Using five complementary short- and long-read sequencing technologies, we phased and assembled >95% of each diploid human genome in a four-generation, 28-member family (CEPH 1463) allowing us to systematically assess de novo mutations (DNMs) and recombination. From this family, we estimate an average of 192 DNMs per generation, including 75.5 de novo single-nucleotide variants (SNVs), 7.4 non-tandem repeat indels, 79.6 de novo indels or structural variants (SVs) originating from tandem repeats, 7.7 centromeric de novo SVs and SNVs, and 12.4 de novo Y chromosome events per generation. STRs and VNTRs are the most mutable with 32 loci exhibiting recurrent mutation through the generations. We accurately assemble 288 centromeres and six Y chromosomes across the generations, documenting de novo SVs, and demonstrate that the DNM rate varies by an order of magnitude depending on repeat content, length, and sequence identity. We show a strong paternal bias (75-81%) for all forms of germline DNM, yet we estimate that 17% of de novo SNVs are postzygotic in origin with no paternal bias. We place all this variation in the context of a high-resolution recombination map ([~]3.5 kbp breakpoint resolution). We observe a strong maternal recombination bias (1.36 maternal:paternal ratio) with a consistent reduction in the number of crossovers with increasing paternal (r=0.85) and maternal (r=0.65) age. However, we observe no correlation between meiotic crossover locations and de novo SVs, arguing against non-allelic homologous recombination as a predominant mechanism. The use of multiple orthogonal technologies, near-telomere-to-telomere phased genome assemblies, and a multi-generation family to assess transmission has created the most comprehensive, publicly available "truth set" of all classes of genomic variants. The resource can be used to test and benchmark new algorithms and technologies to understand the most fundamental processes underlying human genetic variation.

genomics↗

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↗

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↗

bioRxiv2021

STRling: a k-mer counting approach that detects short tandem repeat expansions at known and novel loci

Expansions of short tandem repeats (STRs) cause dozens of rare Mendelian diseases. However, STR expansions, especially those arising from repeats not present in the reference genome, are challenging to detect from short-read sequencing data. Such "novel" STRs include new repeat units occurring at known STR loci, or entirely new STR loci where the sequence is absent from the reference genome. A primary cause of difficulty detecting STR expansions is that reads arising from STR expansions are frequently mismapped or unmapped. To address this challenge, we have developed STRling, a new STR detection algorithm that counts k-mers (short DNA sequences of length k) in DNA sequencing reads, to efficiently recover reads that inform the presence and size of STR expansions. As a result, STRling can call expansions at both known and novel STR loci. STRling has a sensitivity of 83% for 14 known STR disease loci, including the novel STRs that cause CANVAS and DBQD2. It is the first method to resolve the position of novel STR expansions to base pair accuracy. Such accuracy is essential to interpreting the consequence of each expansion. STRling has an estimated 0.078 false discovery rate for known pathogenic loci in unaffected individuals and a 0.20 false discovery rate for genome-wide loci in unaffected individuals when using variants called from long-read data as truth. STRling is fast, scalable on cloud computing, open-source, and freely available at https://github.com/quinlan-lab/STRling.

bioinformatics↗

bioRxiv2020

Effective variant filtering and expected candidate variant yield in studies of rare human disease

In studies of families with rare disease, it is common to screen for de novo mutations, as well as recessive or dominant variants that explain the phenotype. However, the filtering strategies and software used to prioritize high-confidence variants vary from study to study. In an effort to establish recommendations for rare disease research, we derive effective guidelines for variant filtering and report the expected number of candidates for de novo dominant and recessive modes of inheritance. The filters are applied to common attributes, including genotype quality, sequencing depth, allele balance, and population allele frequency. The resulting guidelines yield approximately 10 candidate SNP and INDEL variants per exome, and 19 per genome. For whole genomes, this includes an average of three de novo, ten compound-heterozygotes, one autosomal recessive, four X-linked variants, and roughly 100 candidate variants following autosomal dominant inheritance. The slivar software we developed to establish and rapidly apply these filters to VCF files is available at https://github.com/brentp/slivar under an MIT license, and includes documentation and recommendations for best practices for rare disease analysis.

bioinformatics↗

bioRxiv2019

Pooled-parent exome sequencing to prioritise de novo variants in genetic disease

In the clinical setting, exome sequencing has become standard-of-care in diagnosing rare genetic disorders, however many patients remain unsolved. Trio sequencing has been demonstrated to produce a higher diagnostic yield than singleton (proband-only) sequencing. Parental sequencing is especially useful when a disease is suspected to be caused by a de novo variant in the proband, because parental data provide a strong filter for the majority of variants that are shared by the proband and their parents. However the additional cost of sequencing the parents makes the trio strategy uneconomical for many clinical situations. With two thirds of the sequencing budget being spent on parents, these are funds that could be used to sequence more probands. For this reason many clinics are reluctant to sequence parents.\n\nHere we propose a pooled-parent strategy for exome sequencing of individuals with likely de novo disease. In this strategy, DNA from all the parents of a cohort of unrelated probands is pooled together into a single exome capture and sequencing run. Variants called in the proband can then be filtered if they are also found in the parent pool, resulting in a shorter list of prioritised variants. To evaluate the pooled-parent strategy we performed a series of simulations by combining reads from individual exomes to imitate sample pooling. We assessed the recall and false positive rate and investigated the trade-off between pool size and recall rate. We compared the performance of GATK HaplotypeCaller individual and joint calling, and FreeBayes to genotype pooled samples. Finally, we applied a pooled-parent strategy to a set of real unsolved cases and showed that the parent pool is a powerful filter that is complementary to other commonly used variant filters such as population variant frequencies.

bioinformatics↗

bioRxiv2017

STRetch: detecting and discovering pathogenic short tandem repeats expansions

Short tandem repeat (STR) expansions have been identified as the causal DNA mutation in dozens of Mendelian diseases. Historically, pathogenic STR expansions could only be detected by single locus techniques, such as PCR and electrophoresis. The ability to use short read sequencing data to screen for STR expansions has the potential to reduce both the time and cost to reaching diagnosis and enable the discovery of new causal STR loci. Most existing tools detect STR variation within the read length, and so are unable to detect the majority of pathogenic expansions. Those tools that can detect large expansions are limited to a set of known disease loci and as yet no new disease causing STR expansions have been identified with high-throughput sequencing technologies.\n\nHere we address this by presenting STRetch, a new genome-wide method to detect STR expansions at all loci across the human genome. We demonstrate the use of STRetch for detecting pathogenic STR expansions in short-read whole genome sequencing data with a very low false discovery rate. We further demonstrate the application of STRetch to solve cases of patients with undiagnosed disease and apply STRetch to the analysis of 97 whole genomes to reveal variation at STR loci. STRetch assesses expansions at all STR loci in the genome and allows screening for novel disease-causing STRs.\n\nSTRetch is open source software, available from github.com/Oshlack/STRetch.

bioinformatics↗