Long read sequencing of 3,622 Icelanders provides insight into the role of structural variants in human diseases and other traits

Beyter, Doruk; Ingimundardóttir, Helga; Björnsson, Eyþór; Kristmundsdóttir, Snædís; Mehringer, Svenja; Hardarson, Marteinn T.; Magnúsdóttir, Droplaug N; Kristjansson, Ragnar P.; Gudjonsson, Sigurjon A.; Sverrisson, Sverrir T.; Holley, Guillaume; Eyjolfsson, Gudmundur I.; Ólafsson, Ísleifur; Sigurðardóttir, Ólöf; Másson, Gísli; Þorsteinsdóttir, Unnur; Guðbjartsson, Daníel F.; Sulem, Patrick; Magnússon, Ólafur Þ.; Halldórsson, Bjarni V.; Stefánsson, Kāri

doi:10.1101/848366

Cited by 55 publications

(74 citation statements)

References 80 publications

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“…Recent studies indicated that large structural variants can be identified accurately from genome graphs [32,[53][54][55]. Eventually, a bovine genome graph that unifies multiple breed-specific haplotype-resolved genome assemblies and their sites of variation might provide access to sources of variation that are currently neglected when short sequencing reads are aligned to a linear reference sequence [56,57].…”

Section: Discussionmentioning

confidence: 99%

Bovine breed-specific augmented reference graphs facilitate accurate sequence read mapping and unbiased variant discovery

Crysnanto

Pausch

2019

Preprint

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BackgroundThe current bovine genomic reference sequence was assembled from the DNA of a Hereford cow.The resulting linear assembly lacks diversity because it does not contain allelic variation. Lack of diversity is a drawback of linear references that causes reference allele bias. High nucleotide diversity and the separation of individuals by hundreds of breeds make cattle ideally suited to investigate the optimal composition of variation-aware references. ResultsWe augment the bovine linear reference sequence (ARS-UCD1.2) with variants filtered for allele frequency in dairy (Brown Swiss, Holstein) and dual-purpose (Fleckvieh, Original Braunvieh) cattle breeds to construct either breed-specific or pan-genome reference graphs using the vg toolkit. We find that read mapping is more accurate to variation-aware than linear references if pre-selected variants are used to construct the genome graphs. Graphs that contain random variants do not improve read mapping over the linear reference sequence. Breed-specific augmented and pangenome graphs enable almost similar mapping accuracy improvements over the linear reference.We construct a whole-genome graph that contains the Hereford-based reference sequence and 14 million alleles that have alternate allele frequency greater than 0.03 in the Brown Swiss cattle breed.We show that our novel variation-aware reference facilitates accurate read mapping and unbiased sequence variant genotyping for SNPs and Indels. ConclusionsWe developed the first variation-aware reference graph for an agricultural animal: https://doi.org/10.5281/zenodo.3570312. Our novel reference structure improves sequence read mapping and variant genotyping over the linear reference. Our work is a first step towards the transition from linear to variation-aware reference structures in species with high genetic diversity and many sub-populations.

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Section: Discussionmentioning

confidence: 99%

Bovine breed-specific augmented reference graphs facilitate accurate sequence read mapping and unbiased variant discovery

Crysnanto

Pausch

2019

Preprint

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“…Moreover, the low throughput of modern lrWGS platforms renders them impractical for adoption in most large-scale population studies. The largest published assembly-based PacBio study has analyzed just 15 genomes, 22 while a recent study from Iceland analyzed 1,817 ONT genomes, 24 by comparison to millions of genomes that have already been sequenced or commissioned using srWGS. Given this predominance of srWGS in the current landscape of genomics research, we present here a series of analyses from the HGSVC to: (i) define and quantify the limitations of SV detection from srWGS; (ii) benchmark expectations for the number and class of variants that can be reliably detected from srWGS; (iii) predict the genomic features that drive false positive and false negative discoveries for each technology; and (iv) establish the scientific and clinical advances offered by state-of-the-art lrWGS assembly as a complementary approach to srWGS.…”

Section: Main Textmentioning

confidence: 99%

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

Zhao

Collins

Lee

et al. 2020

Preprint

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AbstractVirtually 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.

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“…After a detailed quality control analysis ( Figure S1) 83,486 SVs were identified, consistent with previous reports using LR-WGS ( Figure S2). 11 Focusing on rare variants (allele count <= 10 in gnomAD v3, NIHR BioResource and NGC project) 14; 17; 18 in SERPINC1 and flanking regions, 10 candidate heterozygous SVs were observed in 9 individuals ( Figure 1C). Visual inspection of read alignments identified an additional heterozygous SV in a region of low coverage involving SERPINC1.…”

Section: Main Textmentioning

confidence: 99%

Long-read sequencing resolves structural variants inSERPINC1causing antithrombin deficiency and identifies a complex rearrangement and a retrotransposon insertion not characterized by routine diagnostic methods

Morena-Barrio

Stephens

Morena‐Barrio

et al. 2020

Preprint

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The identification and characterization of structural variants (SVs) in clinical genetics have remained historically challenging as routine genetic diagnostic techniques have limited ability to evaluate repetitive regions and SVs. Long-read whole-genome sequencing (LR-WGS) has emerged as a powerful approach to resolve SVs. Here, we used LR-WGS to study 19 unrelated cases with type I Antithrombin Deficiency (ATD), the most severe thrombophilia, where routine molecular tests were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs and resolved 10 cases. For the first time, we identified a germline complex rearrangement involved in ATD previously misclassified as a deletion. Additionally, we provided molecular diagnoses for two unresolved individuals that harbored a novel SINE-VNTR-Alu retroelement insertion that we fully characterized by de novo assembly and confirmed by PCR amplification in all affected relatives. Finally, the nucleotide-level resolution achieved for all the SVs allowed breakpoint analysis, which revealed a replication-based mechanism for most of the cases. Our study underscores the utility of LR-WGS as a complementary diagnostic method to identify, characterize, and unveil the molecular mechanism of formation of disease-causing SVs, and facilitates decision making about long-term thromboprophylaxis in ATD patients.

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Long read sequencing of 3,622 Icelanders provides insight into the role of structural variants in human diseases and other traits

Cited by 55 publications

References 80 publications

Bovine breed-specific augmented reference graphs facilitate accurate sequence read mapping and unbiased variant discovery

Bovine breed-specific augmented reference graphs facilitate accurate sequence read mapping and unbiased variant discovery

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

Long-read sequencing resolves structural variants inSERPINC1causing antithrombin deficiency and identifies a complex rearrangement and a retrotransposon insertion not characterized by routine diagnostic methods

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