Agilent cytogenomics v 3

Genome-wide copy number profiling was performed by array-CGH using the SurePrint G3 Human CGH Microarray (Agilent, Santa Clara, CA, USA) according to our previous protocol for FFPE samples [105 (link)]. DNA isolated from peripheral blood from multiple normal individuals was used as control (reference) DNA. Control and case samples were directly labeled using the Bioprimer a-CGH Genomic Labeling kit and hybridized to the arrays for 40 hours. The arrays were scanned using Scanner Agilent G2565CA, and the data extracted using Feature Extraction (FE) software v10.10 (Agilent Tech. Inc.). The Agilent Cytogenomics v.3.0 software (Agilent Technologies Inc., Santa Clara, CA, USA), was used to analyze the data, using the algorithm ADM-2, threshold of 6.0 and an aberration filter with a minimum of 3 probes. Gene amplifications and deletions were defined as minimum average absolute log2 ratio (intensity of the Cy5 dye (reference DNA)/intensity of the Cy3 dye (test DNA) value of >0.25 and <-0.25, respectively, as per Agilent Cytogenomics guidelines. The number of “calls” (total significant number of CNAs) and the specifically affected cytobands were obtained from the generated aberration interval base reports (Agilent Cytogenomics v.3.0).

High resolution array comparative genomic hybridization (a-CGH) analysis was performed on genomic blood DNA of P1, P2, and their parents, using the SurePrint G3 Human CGH Microarray Kit 2 × 400 K in accordance with the manufacturer's instructions (Agilent Technologies, Palo Alto, CA). Data extraction and analysis were performed using Agilent CytoGenomics v.3.0 (Agilent Technologies, Palo Alto, CA). Detected copy number variants (CNVs) were classified according to guidelines reported by Miller et al. (14 (link)) and by the American College of Medical Genetics (15 (link)).