Agilent human genome cgh microarray 60k kit

Comparative genomic hybridization array (aCGH) analysis was performed using Agilent Human Genome CGH Microarray 60K kit (Agilent Technologies, Palo Alto, CA, USA), following the manufacturer’s instructions. A sex-matched commercial DNA sample (Male, Promega, Milan, Italy) was used as reference DNA. Hybridization signals were analyzed using Feature Extraction software (v10.7) and DNA Analytics software (v5.0, Agilent Technologies, Palo Alto, CA, USA). Aberration Detection Method 2 (ADM2) algorithm (threshold 5.0) was used to identify DNA copy number aberrations. We applied a filtering option of a minimum of three aberrant consecutive probes (Wu et al., 2007 (link)) and a minimum absolute average log 2 ratio of 0.30. University of California Santa Cruz (UCSC) human genome assembly hg18 was used as a reference and copy number variations (CNVs) were identified with a database integrated into the Agilent Genomic Workbench analytic software. Log two ratios lower than −0.30 were classified as losses, those greater than 0.30 as gains. The analysis revealed CNVs for the one iPSC clone, consisting of amplification of 36 kb and 175 Kb, on chromosomes 2 and 3, respectively, involving genes with no significant associated clinical phenotype. The clone from healthy control did not reveal any CNVs (Table 5).

For each sample, 2 μg of genomic DNA was prepared following the manufacturers protocol for the Agilent Human Genome CGH microarray 60 K kit (SurePrint G3 Human CGH 8x60K Microarray Kit) or 180 K kit (SurePrint G3 Human CGH 4×180K Microarray Kit) (Agilent Technologies Inc, Santa Clara, CA, USA). This validated aCGH procedure can achieve 99% sensitivity and 99% specificity with an analytical resolution determined by a sliding window of five to seven contiguous oligonucleotides [12 (link)]. The base pair designation was based on the NCBI36/hg18 assembly of the UCSC Human Genome browser (http://genome.ucsc.edu/).