Design of Large‐Insert Jumping Libraries for Structural Variant Detection Using Illumina Sequencing

Hanscom, Carrie; Talkowski, Michael E.

doi:10.1002/0471142905.hg0722s80

Cited by 28 publications

(37 citation statements)

References 10 publications

(14 reference statements)

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“…Customized sequencing libraries were constructed based on published protocols [16] and sequenced with paired-end 50-bp reads on an Illumina HiSeq2500. Library barcodes were demultiplexed with CASAVA v1.7.…”

Section: Methodsmentioning

confidence: 99%

Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars

Macakova

Bohuslavová²,

Vochozková³

et al. 2016

Neurodegener Dis

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Background: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. Objective: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. Methods: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. Results: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. Conclusions: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.

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Section: Methodsmentioning

confidence: 99%

Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars

Macakova

Bohuslavová²,

Vochozková³

et al. 2016

Neurodegener Dis

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“…According to array CGH, complex translocation breakpoints in EGL312 and EGL356 border repetitive regions, so we performed Nextera mate-pair sequencing (Illumina) of 5-to 7-kb inserts. This approach is ideal for junctions in repetitive DNA because mate pairs span repeats and map to unique sequence (Kloosterman et al 2011;Talkowski et al 2011Talkowski et al , 2012Hanscom and Talkowski 2014). We identified discordant reads for one of two junctions expected in EGL312 and for three of four junctions expected in EGL356.…”

Section: Complex Translocations Between Two Chromosomesmentioning

confidence: 96%

Unbalanced translocations arise from diverse mutational mechanisms including chromothripsis

2015

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Unbalanced translocations are a relatively common type of copy number variation and a major contributor to neurodevelopmental disorders. We analyzed the breakpoints of 57 unique unbalanced translocations to investigate the mechanisms of how they form. Fifty-one are simple unbalanced translocations between two different chromosome ends, and six rearrangements have more than three breakpoints involving two to five chromosomes. Sequencing 37 breakpoint junctions revealed that simple translocations have between 0 and 4 base pairs (bp) of microhomology (n = 26), short inserted sequences (n = 8), or paralogous repeats (n = 3) at the junctions, indicating that translocations do not arise primarily from nonallelic homologous recombination but instead form most often via nonhomologous end joining or microhomology-mediated break-induced replication. Three simple translocations fuse genes that are predicted to produce in-frame transcripts of SIRPG-WWOX, SMOC2-PROX1, and PIEZO2-MTA1, which may lead to gain of function. Three complex translocations have inversions, insertions, and multiple breakpoint junctions between only two chromosomes. Whole-genome sequencing and fluorescence in situ hybridization analysis of two de novo translocations revealed at least 18 and 33 breakpoints involving five different chromosomes. Breakpoint sequencing of one maternally inherited translocation involving four chromosomes uncovered multiple breakpoints with inversions and insertions. All of these breakpoint junctions had 0-4 bp of microhomology consistent with chromothripsis, and both de novo events occurred on paternal alleles. Together with other studies, these data suggest that germline chromothripsis arises in the paternal genome and may be transmitted maternally. Breakpoint sequencing of our large collection of chromosome rearrangements provides a comprehensive analysis of the molecular mechanisms behind translocation formation.

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“…8,[14][15][16][17] We generated jumping libraries according to published protocols with a median insert size of 3.75 kb, and subjects were sequenced to an average of 94.13 haploid physical coverage. 18 We performed analyses by integrating three algorithms that we customized for long-insert libraries: LUMPY, 19 cn.MOPS, 20 and our SV classifier. 8 These computational methods detect both anomalous read pairs that cluster in proximity to SV breakpoints and genomic intervals with aberrant sequencing depth indicating copy gain or loss.…”

mentioning

confidence: 99%

Paired-Duplication Signatures Mark Cryptic Inversions and Other Complex Structural Variation

Brand

Collins

Hanscom

et al. 2015

The American Journal of Human Genetics

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Copy-number variants (CNVs) have been the predominant focus of genetic studies of structural variation, and chromosomal microarray (CMA) for genome-wide CNV detection is the recommended first-tier genetic diagnostic screen in neurodevelopmental disorders. We compared CNVs observed by CMA to the structural variation detected by whole-genome large-insert sequencing in 259 individuals diagnosed with autism spectrum disorder (ASD) from the Simons Simplex Collection. These analyses revealed a diverse landscape of complex duplications in the human genome. One remarkably common class of complex rearrangement, which we term dupINVdup, involves two closely located duplications ("paired duplications") that flank the breakpoints of an inversion. This complex variant class is cryptic to CMA, but we observed it in 8.1% of all subjects. We also detected other paired-duplication signatures and duplication-mediated complex rearrangements in 15.8% of all ASD subjects. Breakpoint analysis showed that the predominant mechanism of formation of these complex duplication-associated variants was microhomology-mediated repair. On the basis of the striking prevalence of dupINVdups in this cohort, we explored the landscape of all inversion variation among the 235 highest-quality libraries and found abundant complexity among these variants: only 39.3% of inversions were canonical, or simple, inversions without additional rearrangement. Collectively, these findings indicate that dupINVdups, as well as other complex duplication-associated rearrangements, represent relatively common sources of genomic variation that is cryptic to population-based microarray and low-depth whole-genome sequencing. They also suggest that paired-duplication signatures detected by CMA warrant further scrutiny in genetic diagnostic testing given that they might mark complex rearrangements of potential clinical relevance.

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Design of Large‐Insert Jumping Libraries for Structural Variant Detection Using Illumina Sequencing

Cited by 28 publications

References 10 publications

Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars

Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars

Unbalanced translocations arise from diverse mutational mechanisms including chromothripsis

Paired-Duplication Signatures Mark Cryptic Inversions and Other Complex Structural Variation

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