Genomic workbench v7

Array Comparative Genomic Hybridization analysis was performed using SurePrint G3 Human CGH plus SNP Microarray following the manufacturer’s instructions (Agilent Technologies, Palo Alto, CA, USA). Genomic DNA was extracted using Wizard Genomic DNA Purification Kit (Promega TM, Mannheim, Germany) or GenElute Blood Genomic DNA kit (Sigma, Darmstadt, Germany), according to the manufacturer’s instructions. DNA concentration was determined on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Berlin, Germany). DNA control reference was provided by Agilent. The array was scanned at 3-µm resolution using Agilent microarray scanner and analyzed using Agilent microarray scanner and analyzed using Feature Extraction v10.7 in order to read scanner image and Genomic Workbench v7.0 (Agilent Technologies, Palo Alto, CA, USA) to analyze copy number variations. Significant chromosomal aberration was determined using the algorithm ADM-2 (threshold, 5.0; absolute minimum average log2 ratio, 0.20; with at least three or more consecutive probe sets; see more detailed in [29 (link)]).

Array Comparative Genomic Hybridization (Array CGH) analysis, was performed using SurePrint G3 Human Microarray kit 2x400 K (for 7qs case) and SurePrint G3 Human CGHplusSNP Microarray 4x180 K (for 17ps case) following the manufacturer’s instructions (Agilent Technologies, Palo Alto, CA, USA). Genomic DNA from peripheral blood samples from patients and their parents was extracted using Wizard Genomic DNA Purification Kit (PromegaTM, Mannheim, Germany) according to the manufacturer’s instructions. DNA concentration was determined on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Berlin, Germany).
The array was scanned at 2-µm/3-µm resolution using Agilent microarray scanner and analyzed using Feature Extraction v10.7 and Genomic Workbench v7.0 (Agilent Technologies) in order to read scanner image and to calculate copy number variations. Significant chromosomal aberration was determined using the algorithm ADM-2 (threshold, 5.0; absolute minimum average log2 ratio, 0.20; with at least three or more consecutive probe sets; see more detailed in [35 (link)]).

The Cy5/Cy3 intensity ratios for each spot were converted into log2 ratios. Aberrant chromosome intervals (except for X chromosome) were selected by using Agilent Genomic Workbench v. 7.0. Copy number gain was defined as a log 2 ratio >0.25 and a copy number loss was defined as a log 2 ratio <−0.25. High-level gain or amplification were defined as a log 2 ratio >1 and 1.5 respectively.
Chromosomal locations were defined in terms of their Megabase (Mb) position. For comparison of genomic imbalances shared between human and dog, orthologous regions on human chromosomes were identified using the Ensemble Genome Browser (http://www.ensembl.org/index.html).
In order to find biological ontologies and pathways enriched in recurrent CNAs, functional annotations of all genes mapping to those aberrant regions was performed using the Functional Annotation Tool Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources 6.7, NIAID/NIH (http://david.abcc.ncifcrf.gov/summary.jsp), server “Biological process” (BP), “Molecular function” (MF) and “Cellular component” (CC) annotations were performed by setting gene count = 3 and ease = 0.05. The same parameters were employed also for “Kyoto encyclopedia gene and genome” (KEGG) pathway analysis.