Neurogenetic Variant Analysis by Optical Genome Mapping for Structural Variation Detection-Balanced Genomic Rearrangements, Copy Number Variants, and Repeat Expansions/Contractions

Barseghyan, Hayk; Pang, Andy Wing Chun; Zhang, Yang; Sahajpal, Nikhil; Delpu, Yannick; Lai, Chi-Yu; Lee, Joyce; Tessereau, Chloe; Oldakowski, Mark; Kolhe, Ravindra; Houlden, Henry; Nagy, Péter L.; Bossler, Aaron D.; Chaubey, Alka; Hastie, Alex

doi:10.1007/978-1-0716-2357-2_9

Cited by 3 publications

(6 citation statements)

References 47 publications

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“…Since some repeat expansion disorders have overlapping phenotypic characteristics and strong heterogeneity of symptoms, this option of analyzing the entire human genome at once, proves a large benefitand would allow OGM to become a truly generic test for all established expansion disorders for which expansions lead to SVs >500bp or even ~300 bp as shown for the smallest alleles here (DMPK_04). In line with this, 11 additional samples with a repeat expansion in ATXN10 (Morato Torres et al 2022), C9orf72 (Barseghyan et al 2022), FXN, NOP56 or STARD7 were also analyzed successfully (Supplemental Table S3), suggesting indeed that OGM is suited for most known repeat expansion disorders. Finally, if a repeat expansion disorder is suspected, but is not confirmed by OGM, the generated de novo assembly still allows to identify different types of SVs including other insertions and deletions, but also deletions, inversions and translocations.…”

Section: Discussionmentioning

confidence: 71%

“…In line with this, 11 additional samples with a repeat expansion in ATXN10 (Morato Torres et al . 2022) , C9orf72 (Barseghyan et al 2022), FXN , NOP56 or STARD7 were also analyzed successfully ( Supplemental Table S3 ), suggesting indeed that OGM is suited for most known repeat expansion disorders. Finally, if a repeat expansion disorder is suspected, but is not confirmed by OGM, the generated de novo assembly still allows to identify different types of SVs including other insertions and deletions, but also deletions, inversions and translocations.…”

Section: Discussionmentioning

confidence: 95%

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Optical genome mapping enables accurate repeat expansion testing

van der Sanden,

Neveling,

Shukor

et al. 2024

Preprint

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Short tandem repeats (STRs) are amongst the most abundant class of variations in human genomes and are meiotically and mitotically unstable which leads to expansions and contractions. STR expansions are frequently associated with genetic disorders, with the size of expansions often correlating with the severity and age of onset. Therefore, being able to accurately detect the total repeat expansion length and to identify potential somatic repeat instability is important. Current standard of care (SOC) diagnostic assays include laborious repeat-primed PCR-based tests as well as Southern blotting, which are unable to precisely determine long repeat expansions and/or require a separate set-up for each locus. Sequencing-based assays have proven their potential for the genome-wide detection of repeat expansions but have not yet replaced these diagnostic assays due to their inaccuracy to detect long repeat expansions (short-read sequencing) and their costs (long-read sequencing). Here, we tested whether optical genome mapping (OGM) can efficiently and accurately identify the STR length and assess the stability of known repeat expansions. We performed OGM for 85 samples with known clinically relevant repeat expansions in DMPK, CNBP and RFC1, causing myotonic dystrophy type 1 and 2 and cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), respectively. After performing OGM, we applied three different repeat expansion detection workflows, i.e. manual de novo assembly, local guided assembly (local-GA) and molecule distance script of which the latter two were developed as part of this study. The first two workflows estimated the repeat size for each of the two alleles, while the third workflow was used to detect potential somatic instability. The estimated repeat sizes were compared to the repeat sizes reported after the SOC and concordance between the results was determined. All except one known repeat expansions above the pathogenic repeat size threshold were detected by OGM, and allelic differences were distinguishable, either between wildtype and expanded alleles, or two expanded alleles for recessive cases. An apparent strength of OGM over current SOC methods was the more accurate length measurement, especially for very long repeat expansion alleles, with no upper size limit. In addition, OGM enabled the detection of somatic repeat instability, which was detected in 9/30 DMPK, 23/25 CNBP and 4/30 RFC1 samples, leveraging the analysis of intact, native DNA molecules. In conclusion, for tandem repeat expansions larger than ~300 bp, OGM provides an efficient method to identify exact repeat lengths and somatic repeat instability with high confidence across multiple loci simultaneously, enabling the potential to provide a significantly improved and generic genome-wide assay for repeat expansion disorders.

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Section: Discussionmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 95%

Optical genome mapping enables accurate repeat expansion testing

van der Sanden,

Neveling,

Shukor

et al. 2024

Preprint

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“…In the third protocol, we demonstrate a workflow to assess the known repeat expansion loci in ATXN10 (Morato Torres et al, 2022), C9orf72 (Barseghyan et al, 2022), CNBP (van der Sanden et al, 2024), DMPK (Otero et al, 2021), FMR1 (Iqbal et al, 2023), FXN (Yu et al, 2024), NOP56 (Lam et al, 2023), RFC1 (Facchini et al, 2023), and STARD7 (van der Sanden et al, 2024). These were selected because OGM has previously proven to be able to detect expansions of these repeat loci.…”

Section: Manual De Novo Assembly Workflowmentioning

confidence: 99%

“…Overall, this means that all current repeat expansion detection methods still have their own limitations, with the inability to accurately detect the exact repeat length of (very) large repeats and assessing the somatic instability of repeat expansions being the most important diagnostic limitations. Optical genome mapping (OGM) is another long‐read technology, which can be used as a cost‐effective and easy‐to‐use alternative for structural variant (SV) detection (Barseghyan et al., 2022, 2023) and is also capable of detecting STR expansions and contractions (Facchini et al., 2023; Guruju et al., 2023; Iqbal et al., 2023). By scanning ultra‐high‐molecular‐weight (UHMW) DNA that is labeled at CTTAAG sequence motifs and subsequently assembling genomes and mapping the assemblies against a reference genome, OGM can identify different types of SVs, including insertions, duplications, deletions, inversions, and translocations (Mantere et al., 2021; Neveling et al., 2021).…”

Section: Introductionmentioning

confidence: 99%

Optical Genome Mapping for Applications in Repeat Expansion Disorders

van der Sanden,

Neveling,

Pang

et al. 2024

Current Protocols

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Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat‐primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost‐effective and easy‐to‐use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild‐type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Genomic ultra‐high‐molecular‐weight DNA isolation, labeling, and stainingBasic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® SystemBasic Protocol 3: Manual De Novo Assembly workflowBasic Protocol 4: Local guided assembly workflowBasic Protocol 5: EnFocus Fragile X workflowBasic Protocol 6: Molecule distance script workflow

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“…Amplification-free targeted sequencing and optical genome mapping (OGM) have been used to confirm somatic mosaicism in patient-derived DNA, containing the ancestral motif and mutant ATCCT or ATTCC [ 52 ]. Indeed, OGM is emerging as a powerful platform to delineate complex structural variation, including repeat expansions [ 53 ]. However, current clinical diagnostic testing protocols for SCA10 do not utilise these technologies, instead opting for fragment analysis via capillary array electrophoresis and Southern blot.…”

Section: Pentanucleotide Repeatsmentioning

confidence: 99%

Challenges facing repeat expansion identification, characterisation, and the pathway to discovery

Read,

Davies,

Thompson

et al. 2023

Emerging Topics in Life Sciences

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Tandem repeat DNA sequences constitute a significant proportion of the human genome. While previously considered to be functionally inert, these sequences are now broadly accepted as important contributors to genetic diversity. However, the polymorphic nature of these sequences can lead to expansion beyond a gene-specific threshold, causing disease. More than 50 pathogenic repeat expansions have been identified to date, many of which have been discovered in the last decade as a result of advances in sequencing technologies and associated bioinformatic tools. Commonly utilised diagnostic platforms including Sanger sequencing, capillary array electrophoresis, and Southern blot are generally low throughput and are often unable to accurately determine repeat size, composition, and epigenetic signature, which are important when characterising repeat expansions. The rapid advances in bioinformatic tools designed specifically to interrogate short-read sequencing and the development of long-read single molecule sequencing is enabling a new generation of high throughput testing for repeat expansion disorders. In this review, we discuss some of the challenges surrounding the identification and characterisation of disease-causing repeat expansions and the technological advances that are poised to translate the promise of genomic medicine to individuals and families affected by these disorders.

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Neurogenetic Variant Analysis by Optical Genome Mapping for Structural Variation Detection-Balanced Genomic Rearrangements, Copy Number Variants, and Repeat Expansions/Contractions

Cited by 3 publications

References 47 publications

Optical genome mapping enables accurate repeat expansion testing

Optical genome mapping enables accurate repeat expansion testing

Optical Genome Mapping for Applications in Repeat Expansion Disorders

Challenges facing repeat expansion identification, characterisation, and the pathway to discovery

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