Genome-wide Copy Number Analysis

aCGH was performed using SurePrint G3 Human CGH 4 × 180 K Microarrays (Agilent Technologies, Inc., Santa Clara, CA, USA). Labeling and hybridization were performed according to the manufacturer's instructions. The dual-colored fluorescence signals were scanned using the Agilent Microarray Scanner and translated to log10 ratios using Feature Extraction software (Ver-11.0.1.1, Agilent). Purified DNA was labeled with Cy5-dUTP following the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol (Ver-7.3, Agilent). Data were obtained using Agilent Feature Extraction software 9 and analyzed with Agilent CGH Analytics Version 6.5 software, using the ADM-2 statistical algorithms with 6.0 sensitivity thresholds. Based on the aCGH data, the extracted signals were normalized to log2 ratios using the limma package [57 (link)]. We classified the segmented results into copy losses when the log2 ratios were lower than −0.25 and into copy gains when these were greater than 0.25.