Sequencing of long stretches of repetitive DNA

Bustos, Alfredo de; Cuadrado, Ángeles; Jouve, N.

doi:10.1038/srep36665

Cited by 42 publications

(31 citation statements)

References 23 publications

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“…We do, however, acknowledge the possibility that CHEF-based measurements may be subject to electrophoretic artifacts. However, previous reports find either no or only minimal effect of GC content or repetitiveness on mobility in an agarose gel (Schaffer and Sederoff 1981, Elder and Southern 1983, Pourcel et al 2011, De Bustos et al 2016, giving us confidence in the accuracy of CHEF-based copy number estimates.…”

Section: Discussionmentioning

confidence: 81%

Challenges and Approaches to Genotyping Repetitive DNA

Morton¹,

Hall

Kwan³

et al. 2020

G3 Genes|Genomes|Genetics

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Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.

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Section: Discussionmentioning

confidence: 81%

Challenges and Approaches to Genotyping Repetitive DNA

Morton¹,

Hall

Kwan³

et al. 2020

G3 Genes|Genomes|Genetics

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“…Compared to the constitutive RPGR isoform that spans exons 1 to 19, the ORF15 isoform terminates in intron 15, a GA-rich region that encodes Glu-Gly acidic domains [26]. GArich regions, as with long repeats of other di-and trinucleotides, act as a primary algorithmic challenge in sequence assembling, as the sequence reads lack the capacity to span long repetitive elements [27,28]. Consistently, failures to assemble these structures have been attributable to the gaps in the human genome [29][30][31].…”

Section: Discussionmentioning

confidence: 99%

Fundoscopy-directed genetic testing to re-evaluate negative whole exome sequencing results

Cho

Carvalho

Tanaka

et al. 2020

Orphanet J Rare Dis

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Background: Whole exome sequencing (WES) allows for an unbiased search of the genetic cause of a disease. Employing it as a first-tier genetic testing can be favored due to the associated lower incremental cost per diagnosis compared to when using it later in the diagnostic pathway. However, there are technical limitations of WES that can lead to inaccurate negative variant callings. Our study presents these limitations through a re-evaluation of negative WES results using subsequent tests primarily driven by fundoscopic findings. These tests included targeted gene testing, inherited retinal gene panels, whole genome sequencing (WGS), and array comparative genomic hybridization. Results: Subsequent genetic testing guided by fundoscopy findings identified the following variant types causing retinitis pigmentosa that were not detected by WES: frameshift deletion and nonsense variants in the RPGR gene, 353bp Alu repeat insertions in the MAK gene, and large exonic deletion variants in the EYS and PRPF31 genes. Deep intronic variants in the ABCA4 gene causing Stargardt disease and the GUCY2D gene causing Leber congenital amaurosis were also identified. Conclusions: Negative WES analyses inconsistent with the phenotype should raise clinical suspicion. Subsequent genetic testing may detect genetic variants missed by WES and can make patients eligible for gene replacement therapy and upcoming clinical trials. When phenotypic findings support a genetic etiology, negative WES results should be followed by targeted gene sequencing, array based approach or whole genome sequencing.

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“…Most oomycete genomes sequenced to date were found to contain long repeat regions [80] that cannot be resolved using only a short read technology such as Illumina. Long reads can potentially sequences over these long stretches of repeat sequences, and contribute to the contiguity of the genome assembly [81]. Therefore, the data was complemented with long read PacBio sequences in an attempt to close the gaps between the contigs.…”

Section: Discussionmentioning

confidence: 99%

Genome reconstruction of the non-culturable spinach downy mildew Peronospora effusa by metagenome filtering

Klein

Neilen

Verk

et al. 2019

Preprint

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Peronospora effusa (previously known as P. farinosa f. sp. spinaciae, and here referred to as Pfs) is an obligate biotrophic oomycete that causes downy mildew on spinach (Spinacia oleracea). To combat this destructive disease resistant cultivars are continually bred. However, new Pfs races rapidly break the employed resistance genes. To get insight into the gene repertoire of Pfs and identify infection-related genes, the genome of the first reference race, Pfs1, was sequenced, assembled, and annotated. Due to the obligate biotrophic nature of this pathogen, material for DNA isolation can only be collected from infected spinach leaves that, however, also contain many other microorganisms. The obtained sequences are, therefore, considered a metagenome. To filter and obtain Pfs sequences we utilized the CAT tool to taxonomically annotate ORFs residing on long sequences of a genome pre-assembly. This study is the first to show that CAT filtering performs well on eukaryotic contigs. Based on the taxonomy, determined on multiple ORFs, contaminating long sequences and corresponding reads were removed. Filtered reads were re-assembled to provide a clean and improved Pfs genome sequence of 32.40 Mbp consisting of 8,635 scaffolds. Transcript sequencing of a range of infection time points aided the prediction of a total of 13,277 gene models, including 99 RXLR(-like) effector, and 14 putative Crinkler genes. Comparative analysis identified common features in the secretomes of different obligate biotrophic oomycetes, regardless of their phylogenetic distance. Their secretomes are generally smaller, compared to hemibiotrophic and necrotrophic oomycete species. We observe a reduction in proteins involved cell wall degradation, in Nep1-like proteins (NLPs), proteins with PAN/apple domains, and host translocated effectors. The genome of Pfs1 will be instrumental in studying downy mildew virulence and for understanding the molecular adaptations by which new isolates break spinach resistance.

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Sequencing of long stretches of repetitive DNA

Cited by 42 publications

References 23 publications

Challenges and Approaches to Genotyping Repetitive DNA

Challenges and Approaches to Genotyping Repetitive DNA

Fundoscopy-directed genetic testing to re-evaluate negative whole exome sequencing results

Genome reconstruction of the non-culturable spinach downy mildew Peronospora effusa by metagenome filtering

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