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.

Characterizing a complex CT-rich haplotype in intron 4 of SNCA using large-scale targeted amplicon long-read sequencing.

Parkinson's disease (PD) is a common neurodegenerative disorder with a significant risk proportion driven by genetics. While much progress has been made, most of the heritability remains unknown. This is in-part because previous genetic studies have focused on the contribution of single nucleotide variants. More complex forms of variation, such as structural variants and tandem repeats, are already associated with several synucleinopathies. However, because more sophisticated sequencing methods are usually required to detect these regions, little is understood regarding their contribution to PD. One example is a polymorphic CT-rich region in intron 4 of the SNCA gene. This haplotype has been suggested to be associated with risk of Lewy Body (LB) pathology in Alzheimer's Disease and SNCA gene expression, but is yet to be investigated in PD. Here, we attempt to resolve this CT-rich haplotype and investigate its role in PD. We performed targeted PacBio HiFi sequencing of the region in 1375 PD cases and 959 controls. We replicate the previously reported associations and a novel association between two PD risk SNVs (rs356182 and rs5019538) and haplotype 4, the largest haplotype. Through quantitative trait locus analyzes we identify a significant haplotype 4 association with alternative CAGE transcriptional start site usage, not leading to significant differential SNCA gene expression in post-mortem frontal cortex brain tissue. Therefore, disease association in this locus might not be biologically driven by this CT-rich repeat region. Our data demonstrates the complexity of this SNCA region and highlights that further follow up functional studies are warranted.

STRchive: a dynamic resource detailing population-level and locus-specific insights at tandem repeat disease loci.

Approximately 3% of the human genome consists of repetitive elements called tandem repeats (TRs), which include short tandem repeats (STRs) of 1-6bp motifs and variable number tandem repeats (VNTRs) of 7+bp motifs. TR variants contribute to several dozen mono- and polygenic diseases but remain understudied and "enigmatic," particularly relative to single nucleotide variants. It remains comparatively challenging to interpret the clinical significance of TR variants. Although existing resources provide portions of necessary data for interpretation at disease-associated loci, it is currently difficult or impossible to efficiently invoke the additional details critical to proper interpretation, such as motif pathogenicity, disease penetrance, and age of onset distributions. It is also often unclear how to apply population information to analyses. We present STRchive (S-T-archive, http://strchive.org/ ), a dynamic resource consolidating information on TR disease loci in humans from research literature, up-to-date clinical resources, and large-scale genomic databases, with the goal of streamlining TR variant interpretation at disease-associated loci. We apply STRchive -including pathogenic thresholds, motif classification, and clinical phenotypes-to a gnomAD cohort of ∼18.5k individuals genotyped at 60 disease-associated loci. Through detailed literature curation, we demonstrate that the majority of TR diseases affect children despite being thought of as adult diseases. Additionally, we show that pathogenic genotypes can be found within gnomAD which do not necessarily overlap with known disease prevalence, and leverage STRchive to interpret locus-specific findings therein. We apply a diagnostic blueprint empowered by STRchive to relevant clinical vignettes, highlighting possible pitfalls in TR variant interpretation. As a living resource, STRchive is maintained by experts, takes community contributions, and will evolve as understanding of TR diseases progresses.

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

The factors driving or preventing pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14 (GAA)·(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a common 5'-flanking variant in 70.34% of alleles analyzed (3,463/4,923) that represents the phylogenetically ancestral allele and is present on all major haplotypes. This common sequence variation is present nearly exclusively on nonpathogenic alleles with fewer than 30 GAA-pure triplets and is associated with enhanced stability of the repeat locus upon intergenerational transmission and increased Fiber-seq chromatin accessibility.

Analysis and benchmarking of small and large genomic variants across tandem repeats.

Tandem repeats (TRs) are highly polymorphic in the human genome, have thousands of associated molecular traits and are linked to over 60 disease phenotypes. However, they are often excluded from at-scale studies because of challenges with variant calling and representation, as well as a lack of a genome-wide standard. Here, to promote the development of TR methods, we created a catalog of TR regions and explored TR properties across 86 haplotype-resolved long-read human assemblies. We curated variants from the Genome in a Bottle (GIAB) HG002 individual to create a TR dataset to benchmark existing and future TR analysis methods. We also present an improved variant comparison method that handles variants greater than 4 bp in length and varying allelic representation. The 8.1% of the genome covered by the TR catalog holds ~24.9% of variants per individual, including 124,728 small and 17,988 large variants for the GIAB HG002 'truth-set' TR benchmark. We demonstrate the utility of this pipeline across short-read and long-read technologies.

Sequence composition changes in short tandem repeats: heterogeneity, detection, mechanisms and clinical implications.

Short tandem repeats (STRs) are a class of repetitive elements, composed of tandem arrays of 1-6 base pair sequence motifs, that comprise a substantial fraction of the human genome. STR expansions can cause a wide range of neurological and neuromuscular conditions, known as repeat expansion disorders, whose age of onset, severity, penetrance and/or clinical phenotype are influenced by the length of the repeats and their sequence composition. The presence of non-canonical motifs, depending on the type, frequency and position within the repeat tract, can alter clinical outcomes by modifying somatic and intergenerational repeat stability, gene expression and mutant transcript-mediated and/or protein-mediated toxicities. Here, we review the diverse structural conformations of repeat expansions, technological advances for the characterization of changes in sequence composition, their clinical correlations and the impact on disease mechanisms.

Characterization and visualization of tandem repeats at genome scale.

Tandem repeat (TR) variation is associated with gene expression changes and numerous rare monogenic diseases. Although long-read sequencing provides accurate full-length sequences and methylation of TRs, there is still a need for computational methods to profile TRs across the genome. Here we introduce the Tandem Repeat Genotyping Tool (TRGT) and an accompanying TR database. TRGT determines the consensus sequences and methylation levels of specified TRs from PacBio HiFi sequencing data. It also reports reads that support each repeat allele. These reads can be subsequently visualized with a companion TR visualization tool. Assessing 937,122 TRs, TRGT showed a Mendelian concordance of 98.38%, allowing a single repeat unit difference. In six samples with known repeat expansions, TRGT detected all expansions while also identifying methylation signals and mosaicism and providing finer repeat length resolution than existing methods. Additionally, we released a database with allele sequences and methylation levels for 937,122 TRs across 100 genomes.

RExPRT: a machine learning tool to predict pathogenicity of tandem repeat loci.

Expansions of tandem repeats (TRs) cause approximately 60 monogenic diseases. We expect that the discovery of additional pathogenic repeat expansions will narrow the diagnostic gap in many diseases. A growing number of TR expansions are being identified, and interpreting them is a challenge. We present RExPRT (Repeat EXpansion Pathogenicity pRediction Tool), a machine learning tool for distinguishing pathogenic from benign TR expansions. Our results demonstrate that an ensemble approach classifies TRs with an average precision of 93% and recall of 83%. RExPRT's high precision will be valuable in large-scale discovery studies, which require prioritization of candidate loci for follow-up studies.

Benchmarking of small and large variants across tandem repeats.

Tandem repeats (TRs) are highly polymorphic in the human genome, have thousands of associated molecular traits, and are linked to over 60 disease phenotypes. However, their complexity often excludes them from at-scale studies due to challenges with variant calling, representation, and lack of a genome-wide standard. To promote TR methods development, we create a comprehensive catalog of TR regions and explore its properties across 86 samples. We then curate variants from the GIAB HG002 individual to create a tandem repeat benchmark. We also present a variant comparison method that handles small and large alleles and varying allelic representation. The 8.1% of the genome covered by the TR catalog holds ∼24.9% of variants per individual, including 124,728 small and 17,988 large variants for the GIAB HG002 TR benchmark. We work with the GIAB community to demonstrate the utility of this benchmark across short and long read technologies.

A deep population reference panel of tandem repeat variation.

Tandem repeats (TRs) represent one of the largest sources of genetic variation in humans and are implicated in a range of phenotypes. Here we present a deep characterization of TR variation based on high coverage whole genome sequencing from 3550 diverse individuals from the 1000 Genomes Project and H3Africa cohorts. We develop a method, EnsembleTR, to integrate genotypes from four separate methods resulting in high-quality genotypes at more than 1.7 million TR loci. Our catalog reveals novel sequence features influencing TR heterozygosity, identifies population-specific trinucleotide expansions, and finds hundreds of novel eQTL signals. Finally, we generate a phased haplotype panel which can be used to impute most TRs from nearby single nucleotide polymorphisms (SNPs) with high accuracy. Overall, the TR genotypes and reference haplotype panel generated here will serve as valuable resources for future genome-wide and population-wide studies of TRs and their role in human phenotypes.

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.

A phenome-wide association study of methylated GC-rich repeats identifies a GCC repeat expansion in AFF3 as a significant cause of intellectual disability.

GC-rich tandem repeat expansions (TREs) are often associated with DNA methylation, gene silencing and folate-sensitive fragile sites and underlie several congenital and late-onset disorders. Through a combination of DNA methylation profiling and tandem repeat genotyping, we identified 24 methylated TREs and investigated their effects on human traits using PheWAS in 168,641 individuals from the UK Biobank, identifying 156 significant TRE:trait associations involving 17 different TREs. Of these, a GCC expansion in the promoter of AFF3 was linked with a 2.4-fold reduced probability of completing secondary education, an effect size comparable to several recurrent pathogenic microdeletions. In a cohort of 6,371 probands with neurodevelopmental problems of suspected genetic etiology, we observed a significant enrichment of AFF3 expansions compared to controls. With a population prevalence that is at least 5-fold higher than the TRE that causes fragile X syndrome, AFF3 expansions represent a significant cause of neurodevelopmental delay.

A deep population reference panel of tandem repeat variation.

Tandem repeats (TRs) represent one of the largest sources of genetic variation in humans and are implicated in a range of phenotypes. Here we present a deep characterization of TR variation based on high coverage whole genome sequencing from 3,550 diverse individuals from the 1000 Genomes Project and H3Africa cohorts. We develop a method, EnsembleTR, to integrate genotypes from four separate methods resulting in high-quality genotypes at more than 1.7 million TR loci. Our catalog reveals novel sequence features influencing TR heterozygosity, identifies population-specific trinucleotide expansions, and finds hundreds of novel eQTL signals. Finally, we generate a phased haplotype panel which can be used to impute most TRs from nearby single nucleotide polymorphisms (SNPs) with high accuracy. Overall, the TR genotypes and reference haplotype panel generated here will serve as valuable resources for future genome-wide and population-wide studies of TRs and their role in human phenotypes.

Recurrent repeat expansions in human cancer genomes.

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3-8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, 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, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.

An intronic GAA repeat expansion in FGF14 causes the autosomal-dominant adult-onset ataxia SCA50/ATX-FGF14.

Adult-onset cerebellar ataxias are a group of neurodegenerative conditions that challenge both genetic discovery and molecular diagnosis. In this study, we identified an intronic (GAA) repeat expansion in fibroblast growth factor 14 (FGF14). Genetic analysis of 95 Australian individuals with adult-onset ataxia identified four (4.2%) with (GAA)>300 and a further nine individuals with (GAA)>250. PCR and long-read sequence analysis revealed these were pure (GAA) repeats. In comparison, no control subjects had (GAA)>300 and only 2/311 control individuals (0.6%) had a pure (GAA)>250. In a German validation cohort, 9/104 (8.7%) of affected individuals had (GAA)>335 and a further six had (GAA)>250, whereas 10/190 (5.3%) control subjects had (GAA)>250 but none were (GAA)>335. The combined data suggest (GAA)>335 are disease causing and fully penetrant (p = 6.0 × 10-8, OR = 72 [95% CI = 4.3-1,227]), while (GAA)>250 is likely pathogenic with reduced penetrance. Affected individuals had an adult-onset, slowly progressive cerebellar ataxia with variable features including vestibular impairment, hyper-reflexia, and autonomic dysfunction. A negative correlation between age at onset and repeat length was observed (R2 = 0.44, p = 0.00045, slope = -0.12) and identification of a shared haplotype in a minority of individuals suggests that the expansion can be inherited or generated de novo during meiotic division. This study demonstrates the power of genome sequencing and advanced bioinformatic tools to identify novel repeat expansions via model-free, genome-wide analysis and identifies SCA50/ATX-FGF14 as a frequent cause of adult-onset ataxia.

REViewer: haplotype-resolved visualization of read alignments in and around tandem repeats.

BACKGROUND: Expansions of short tandem repeats are the cause of many neurogenetic disorders including familial amyotrophic lateral sclerosis, Huntington disease, and many others. Multiple methods have been recently developed that can identify repeat expansions in whole genome or exome sequencing data. Despite the widely recognized need for visual assessment of variant calls in clinical settings, current computational tools lack the ability to produce such visualizations for repeat expansions. Expanded repeats are difficult to visualize because they correspond to large insertions relative to the reference genome and involve many misaligning and ambiguously aligning reads. RESULTS: We implemented REViewer, a computational method for visualization of sequencing data in genomic regions containing long repeat expansions and FlipBook, a companion image viewer designed for manual curation of large collections of REViewer images. To generate a read pileup, REViewer reconstructs local haplotype sequences and distributes reads to these haplotypes in a way that is most consistent with the fragment lengths and evenness of read coverage. To create appropriate training materials for onboarding new users, we performed a concordance study involving 12 scientists involved in short tandem repeat research. We used the results of this study to create a user guide that describes the basic principles of using REViewer as well as a guide to the typical features of read pileups that correspond to low confidence repeat genotype calls. Additionally, we demonstrated that REViewer can be used to annotate clinically relevant repeat interruptions by comparing visual assessment results of 44 FMR1 repeat alleles with the results of triplet repeat primed PCR. For 38 of these alleles, the results of visual assessment were consistent with triplet repeat primed PCR. CONCLUSIONS: Read pileup plots generated by REViewer offer an intuitive way to visualize sequencing data in regions containing long repeat expansions. Laboratories can use REViewer and FlipBook to assess the quality of repeat genotype calls as well as to visually detect interruptions or other imperfections in the repeat sequence and the surrounding flanking regions. REViewer and FlipBook are available under open-source licenses at https://github.com/illumina/REViewer and https://github.com/broadinstitute/flipbook respectively.

STRipy: A graphical application for enhanced genotyping of pathogenic short tandem repeats in sequencing data.

Expansions of short tandem repeats (STRs) have been implicated as the causal variant in over 50 diseases known to date. There are several tools which can genotype STRs from high-throughput sequencing (HTS) data. However, running these tools out of the box only allows around half of the known disease-causing loci to be genotyped. Furthermore, the genotypes estimated at these loci are often underestimated with maximum lengths limited to either the read or fragment length, which is less than the pathogenic cutoff for some diseases. Although analysis tools can be customized to genotype extra loci, this requires proficiency in bioinformatics to set up, limiting their widespread usage by other researchers and clinicians. To address these issues, we have developed a new software called STRipy, which is able to target all known disease-causing STRs from HTS data. We created an intuitive graphical interface for STRipy and significantly simplified the detection of STRs expansions. Moreover, we genotyped all disease loci for over two and half thousand samples to provide population-wide distributions to assist with interpretation of results. We believe the simplicity and breadth of STRipy will increase the genotyping of STRs in sequencing data resulting in further diagnoses of rare STR diseases.

Whole genome sequencing for the diagnosis of neurological repeat expansion disorders in the UK: a retrospective diagnostic accuracy and prospective clinical validation study.

BACKGROUND: Repeat expansion disorders affect about 1 in 3000 individuals and are clinically heterogeneous diseases caused by expansions of short tandem DNA repeats. Genetic testing is often locus-specific, resulting in underdiagnosis of people who have atypical clinical presentations, especially in paediatric patients without a previous positive family history. Whole genome sequencing is increasingly used as a first-line test for other rare genetic disorders, and we aimed to assess its performance in the diagnosis of patients with neurological repeat expansion disorders. METHODS: We retrospectively assessed the diagnostic accuracy of whole genome sequencing to detect the most common repeat expansion loci associated with neurological outcomes (AR, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, C9orf72, CACNA1A, DMPK, FMR1, FXN, HTT, and TBP) using samples obtained within the National Health Service in England from patients who were suspected of having neurological disorders; previous PCR test results were used as the reference standard. The clinical accuracy of whole genome sequencing to detect repeat expansions was prospectively examined in previously genetically tested and undiagnosed patients recruited in 2013-17 to the 100 000 Genomes Project in the UK, who were suspected of having a genetic neurological disorder (familial or early-onset forms of ataxia, neuropathy, spastic paraplegia, dementia, motor neuron disease, parkinsonian movement disorders, intellectual disability, or neuromuscular disorders). If a repeat expansion call was made using whole genome sequencing, PCR was used to confirm the result. FINDINGS: The diagnostic accuracy of whole genome sequencing to detect repeat expansions was evaluated against 793 PCR tests previously performed within the NHS from 404 patients. Whole genome sequencing correctly classified 215 of 221 expanded alleles and 1316 of 1321 non-expanded alleles, showing 97·3% sensitivity (95% CI 94·2-99·0) and 99·6% specificity (99·1-99·9) across the 13 disease-associated loci when compared with PCR test results. In samples from 11 631 patients in the 100 000 Genomes Project, whole genome sequencing identified 81 repeat expansions, which were also tested by PCR: 68 were confirmed as repeat expansions in the full pathogenic range, 11 were non-pathogenic intermediate expansions or premutations, and two were non-expanded repeats (16% false discovery rate). INTERPRETATION: In our study, whole genome sequencing for the detection of repeat expansions showed high sensitivity and specificity, and it led to identification of neurological repeat expansion disorders in previously undiagnosed patients. These findings support implementation of whole genome sequencing in clinical laboratories for diagnosis of patients who have a neurological presentation consistent with a repeat expansion disorder. FUNDING: Medical Research Council, Department of Health and Social Care, National Health Service England, National Institute for Health Research, and Illumina.