Agilent cytogenomics

Genomic DNAs of Guangxi HCC cohort were extracted from fresh tissue using Puregene Kit following manufacturer’s instruction (Qiagen Inc., Valencia, CA). DNA concentration was measured using a NanoDrop spectrophotometer (ND-1000, Thermo Fisher Scientific Inc., Waltham, MA) and high molecular weight DNA quality was verified by agarose gel electrophoresis. For each aCGH analysis, 2.5 μg of test genomic DNA from the patient and 2.5 μg of control DNA from a sex-matched or -mismatched healthy individual were used following the manufacturer’s protocol for the Agilent Human Genome aCGH microarray 60 K (Agilent Technologies Inc., Santa Clara, CA). The differential labeling of test and control DNAs, comparative hybridization onto 8 × 60 K Agilent slides, posthybridization wash, slide scanning, image feature extraction were processed following manufacturer’s instruction. The data were analyzed using Agilent CytoGenomics (version 5.0.2.5) with the built-in ADM-2 algorithm set at threshold value of 6, a cut off value of 0.2, and a filter of ten probes. All CNVs except the recognized copy number variants from the HCC cohort of Genomic Variants (http://projects.tcag.ca/variation/) were recorded. The base pair designations from the Agilent 60 K array are according to the Feb. 2009 (GRCh37/hg19) on the UCSC Human Genome browser (http://genome.ucsc.edu/).

The hybridized chip was scanned, and images were quantified with FEATURE EXTRACTION software (Agilent Technologies). Data were normalized using a vendor-provided equation (log2 [Cy3/Cy5] 0.25). CNVs selection was conducted in Agilent CytoGenomics (Agilent Technologies), followed by a filter to select regions with three or more adjacent probes and a minimum average log2 ratio + 0.25.

Genomic DNA extraction from peripheral blood lymphocytes was carried out with the Jetquick Blood and Cell Culture DNA Midi Spin Kit (Genomed, Löhne, Germany), according to the manufacturer’s instructions. DNA concentration and purity were evaluated using a NanoDrop1000 spectrophotometer. Array-CGH was performed for fine mapping of the 22q11.2 region, using the Agilent SurePrint G3 Human Genome Microarray 4X180K (Agilent Technologies, Santa Clara, USA). Labeling and hybridization were carried out with sex-matched reference DNA by using an Agilent Genomic DNA Enzymatic Labeling Kit (Agilent Technologies, Santa Clara, USA), according to the manufacturer’s instructions. Array slides images were acquired on an Agilent scanner and the data were processed with Feature Extraction software (v10.7). Results were analyzed with Agilent Genomic Workbench (v6.5) and Agilent Cytogenomics (v2.7.7.0), according to Human Genome build 19, and interpreted by databases consultation. Regions of copy number change were interpreted with the aid of the UCSC Genome Browser [31 ].