Computational protocol: Copy number variations in endoglin locus: mapping of large deletions in Spanish families with hereditary hemorrhagic telangiectasia type 1

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Protocol publication

[…] Breakpoints were studied using a custom designed CGH microarray (Agilent) to detect copy number variations. A tiling microarray was designed with eArray (Agilent). The array contains a total of 15,744 probes, including: i) 7,568 probes of ENG; ii) 3,927 probes from the rest of chromosome 9; iii) 1,262 probes included for normalization; and iv) 2,987 probes as Agilent controls. A total of 7,568 probes were selected in the region of interest (chr9: 130548305–130661871, hg19) covering the ENG gene and the surrounding upstream and downstream sequences to complete a total area of 113-Kb, with the ENG gene in its center (Figure  ). Probes were designed with an average probe spacing of 15-bp, and allowed to vary in size (from 45- to 60-mer) in order to maintain their Tm about 80°C. The rest of the chromosome 9 was covered with 3,927 additional probes. For normalization purposes during data analysis, 1,262 probes, distributed among all chromosomes, were also included. 2,987 probes were Agilent controls. A probe performance score was assigned to each probe by the software.Labeling was carried out according to manufacturer protocol l (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, v6.3). Briefly, eight genomic DNA samples, including four controls (non HTT patients), were Cy5 labeled and co-hybridized with a sex matched, Cy3 labeled human genomic reference DNA (Promega). After hybridization, arrays were washed and the signal for each probe was captured (scanned) with an Agilent scanner. Images were analyzed using Agilent Feature Extraction v10.7 software. The quality of the hybridization was estimated by the DLRS parameter, indicating the dispersion among the hybridization signaling of the probes. In all cases, good DLRS values, between 0.1 and 0.2 were obtained.Finally, raw data were analyzed with Agilent Genomic Workbench 6.5. Pre-processing steps were done by applying GC correction and centralization. Probes with scores below 0.5 were filtered out. Aberration calling was done with the ADM-2 algorithm with a threshold of 0.6. An aberration was considered when there were at least 5 consecutives probes with a minimum absolute average log ratio of 0.25. The breakpoint regions were inspected with a UCSC Genome Browser, using the Repeat Masker database to identify repeated elements. […]

Pipeline specifications

Software tools Agilent Feature Extraction, Agilent Genomic Workbench
Databases UCSC Genome Browser
Application Genome data visualization
Organisms Cucumber necrosis virus
Diseases Arteriovenous Malformations, Gastrointestinal Diseases, Telangiectasia, Hereditary Hemorrhagic, Telangiectasis, Vascular Diseases, Genetic Diseases, Inborn