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Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations

ABSTRACT: Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. Genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs. The current application of PET sequencing with short insert size DNA is insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We have developed a robust procedure to generate PET sequencing data using large DNA inserts of 10 - 20 kb for the identification of SVs. We compared the characteristics of the large insert libraries with short insert (1 kb) libraries with the same sequencing depths and costs. Although short insert libraries bear an advantage in identifying small deletions, they do not provide a significantly better breakpoint resolution. Large inserts are superior to short inserts in providing higher physical genome coverage and therefore achieve greater sensitivity for the identification of the different types of SVs, including copy number neutral and complex events. Further, large inserts allow the identification of SVs within repetitive sequences which cannot be spanned by short inserts.

ORGANISM(S): Homo sapiens

PROVIDER: GSE32674 | GEO | 2012/10/04

SECONDARY ACCESSION(S): PRJNA147033

REPOSITORIES: GEO

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Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations

Project description:Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. Genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs. The current application of PET sequencing with short insert size DNA is insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We have developed a robust procedure to generate PET sequencing data using large DNA inserts of 10 - 20 kb for the identification of SVs. We compared the characteristics of the large insert libraries with short insert (1 kb) libraries with the same sequencing depths and costs. Although short insert libraries bear an advantage in identifying small deletions, they do not provide a significantly better breakpoint resolution. Large inserts are superior to short inserts in providing higher physical genome coverage and therefore achieve greater sensitivity for the identification of the different types of SVs, including copy number neutral and complex events. Further, large inserts allow the identification of SVs within repetitive sequences which cannot be spanned by short inserts. Structural variations of three cancer cell lines using short (1 kb) and long (10 kb and 20 kb) insert size DNA fragments

2012-10-04 | E-GEOD-32674 | biostudies-arrayexpress

Comprehensive Long Span Paired-End-Tag Mapping Reveals Characteristic Patterns of Structural Variations in Epithelial Cancer Genomes

Project description:Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long span paired-end-tag (PET) sequencing approach using 10 Kb genomic DNA inserts to study human genome structural variations (SVs). The use of 10 Kb insert size allows the identification of breakpoints within repetitive or homology containing regions of a few Kb in size and results in a higher physical coverage compared to small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and 2 non-cancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germline SVs, whereas tandem duplications, unpaired inversions, inter-chromosomal translocations, and complex rearrangements are overrepresented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA-PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures which are compatible with breakage-fusion-bridge cycles, and discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers. Structural variations of 15 human cancer samples and 2 human normal samples were identified by long span paired-end sequencing

2011-04-06 | E-GEOD-26954 | biostudies-arrayexpress

Comprehensive Long Span Paired-End-Tag Mapping Reveals Characteristic Patterns of Structural Variations in Epithelial Cancer Genomes

2011-04-06 | GSE26954 | GEO

Genome sequencing coupled with iPSC technology identifies GTDC1 as a novel candidate gene involved in Neurodevelopmental Disorders

Project description:We identified genomic structural alterations of six patients with signs of neurodevelopmental disorder (NDDs) that harbour chromosomal rearrangements using large-insert paired-end tag sequencing (DNA-PET). This technique allowed the refinement of chromosomal breakpoints and lead to the identification of seven disrupted genes (GNAQ, RBFOX3, UNC5D, TMEM47, NCAPG2, GTDC1 and XIAP). For one patient we filtered the entire panel of structural variations (SVs) with his parents and identified a unique SV that disrupted a single gene: GTDC1. We then validated the functional consequences of the chromosomal breakpoint disruption of GTDC1 by using patient-derived iPSCs. By differentiating these cells into neural progenitor cells (NPCs) and neurons, we interrogated the disease process at the cellular level and observed defects in the proliferation and glycosylation status of NPCs and also defects in neuronal maturation and function. We compared these results with GTDC1-deficient wild-type human NPCs and neurons, and observed similar phenotypic features as in the patient-derived cells which confirm that GTDC1 is involved in the patient’s phenotype. We show here that the combination of genomic screening with iPSCs technology provides a mechanistic insight into possible contributory effects of candidate genes implicated in NDDs and for personalized medicine. Structural variations were identified by long insert DNA paired-end tag (DNA-PET) sequencing, a mate-pair sequencing approach.

2014-09-03 | E-GEOD-51430 | biostudies-arrayexpress

Genome sequencing coupled with iPSC technology identifies GTDC1 as a novel candidate gene involved in Neurodevelopmental Disorders

2014-09-03 | GSE51430 | GEO

Clostridium acetobutylicum ATCC 824 (pLib1) butanol stresses with repeated transfers

Project description:Metabolite accumulation has pleiotropic, including toxic, effects on cellular physiology, with a variety of genetic resistance mechanisms previously identified. Using random genomic libraries and DNA microarrays, library inserts were preferentially enriched by culturing in media containing butanol, with successive transfers into fresh media containing incrementally increasing butanol concentrations. Keywords: genomic library preferential enrichment

2007-12-29 | GSE6289 | GEO

Comparative genomic hybridization of plasmids containing genomic library clones grown in E. coli to monitor copy number changes over time

Project description:We report a genome-wide, multi-scale approach to simultaneously measure the effect that the increased copy of each gene and/or operon has on a desired trait or phenotype. The method involves i) growth selections on a mixture of several different plasmid based genomic libraries of defined insert sizes or SCALEs, ii) micro-array studies of enriched plasmid DNA, and a ii) mathematical multi-scale analysis (MSA) that precisely identifies the relevant genetic elements. This approach allows for identification of all single open reading frames and larger multi-gene fragments within a genomic library that alter the expression of a given phenotype. We have demonstrated this method in E. coli by monitoring, in parallel, a population of >106 genomic library clones of different insert sizes, throughout continuous selections over a period of 100 generations.

2006-10-26 | E-TABM-143 | biostudies-arrayexpress

Homo sapiens isolate:HG00733

Project description:Sequel Large Insert Library Genomic Sequence of HG00733

| PRJNA483067 | ENA

Lenti-miR Library WiDR, Pre vs Post Intrahepatic Selection

Project description:Cells were transduced with a lentiviral Lenti-miR library composed of 661 miRNAs (System Biosciences) at a low multiplicity of infection (MOI) such that each cell over-expressed a single miRNA. The transduced population was then injected intra-hepatically into NOD-SCID mice for in vivo selection of miRNAs that when over-expressed, either promoted or suppressed metastatic liver colonization. Genomic DNA PCR amplication and recovery of lenti-viral miRNA inserts was performed on cells prior to injection, and from liver nodules, according to the manufacturer’s protocol. miRNA array profiling allowed for miRNA insert quantification prior to and subsequent to in vivo selection.

2015-01-16 | GSE56319 | GEO

Lenti-miR Library SW620, Pre vs Post Intrahepatic Selection

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