Oligonucleotide array based cgh for genomic dna analysis

Purified DNA from patient-derived tumor samples was labeled with Cy5-dUTP following the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol (Ver-7.3, Agilent). Cy5-labeled DNA was quantified together with reference DNA samples labeled with Cy3-dUTP to determine the DNA concentration using an ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE, USA). Labeled test and reference samples were then hybridized to SurePrint G3 Human CGH 4 × 180 K Microarrays (Agilent) according to the manufacturer’s standard protocol. 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).

aCGH analyses on oligonucleotide arrays were performed according to the manufacturer’s instructions (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, Version 6.4, August 2011, G4410-90010) with slight modifications as described in [42 (link)]. All CGH arrays were processed using the Microarray Scanner G2565CA by Agilent Technologies with 3 μm resolution and 16 bit color depth. The output image files were imported, normalized and fluorescent ratios for each probe were determined using Feature Extraction software (Agilent Technologies, Version 10.7.3.1, Protocol CGH_107_Sep09). Feature Extraction output files were imported into the Genomic Workbench 5.0.14 software. aCGH data were examined using the aberration detection method 2 (ADM-2) algorithm with a threshold of 6.0. No centralization was applied. An aberration filter was defined for identifying copy number alterations, where changes only were considered as true positive events with a minimum log2ratio of 0.3 and a minimum of 50 consecutive probes with the same polarity per region.

Array CGH was performed on oligonucleotide-based SurePrint G3 Human CGH 4×180K microarray slides (design code: 022060) according the protocol provided by the manufacturer (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, version 7.1, December 2011). Slight modifications were introduced WGA products processed with the PCR-based labeling approaches. Here, the hybridization mix consisted of 5.0 µg of Cot1-DNA (Roche Diagnostics), 12 µl of 10x Blocking Reagent (Agilent Technologies), 60 µl of 2x Hi RPM Hybridization Buffer, 1% (v/v) of both Tween20 and Igepal and 19 µl of both test and reference DNA. For each hybridization 100 µl of the hybridization mix was applied on the array and hybridized at 65°C for 24 h. Following the hybridization, the slides were washed twice for 2:30 min in Oligo aCGH/ChIP-on-Chip Wash Buffer 1 (Agilent Technologies) at room temperature, twice for 30 sec min in Oligo aCGH/ChIP-on-Chip Wash Buffer 2 (Agilent Technologies) at 37°C. Washed slides were immersed in acetonitrile to remove all remaining traces of the wash buffers. Finally, slides were scanned using an Agilent Microarray Scanner Type C.

DNA from cell lines was isolated using QIAamp DNA mini kit (Qiagen). For the healthy diploid reference, 1.5 μg genomic DNA was isolated from the PBMCs of a healthy female donor using QIAamp^® DNA Mini Kit (Qiagen). DNA was fragmented, labeled, purified, and hybridized to Agilent 2 × 10⁵ K arrays according to the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis (version 6.2.1). Washing and scanning in Agilent BioScanner B took place immediately after hybridization. Data was extracted using Agilent’s Feature Extraction Software. The featured extracted data from the 15 cell lines was analysed using Agilent CytoGenomics Software. Aberrations were called using the ADM-2 default algorithm using a threshold of 6.0.

To perform CGH arrays, we used genomic DNA from ENU-treated iPSC-derived EBs and Bl-CFCs compared to CML-iPSCs without ENU treatment. We followed Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol. Briefly, we performed digestion from 500 ng of DNA with SureTag DNA labeling kit (4-packs). After DNA amplification, CML-iPSC was labeled with Cyanine 3 and the tested samples with Cyanine 5. Assembled chambers were loaded into the oven rotator rack; hybridizations were performed at 67 °C for 24 h at 20 rpm.

Array CGH was performed using Agilent Human Genome Unrestricted G3 ISCA v2 Sureprint 8 × 60K oligo-array (Amadid 021924) (Agilent, Santa Clara, CA) (Kallioniemi et al., 1992 (link)). DNA was isolated from peripheral blood leukocytes using the NucleoSpin^®Dx Blood DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA) as recommended by the manufacturer. For calculation of the concentration and purity of the isolated DNA NanoDrop spectrophotometer was used. Labeling and hybridization of the samples was made according to the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis—Enzymatic Labeling Protocol. Washing was performed following the instructions of Agilent Protocol v7.2. The results were obtained by Agilent dual laser scanner G2565CA and processed with Agilent Feature Extraction software (v10.10.1.1.). Agilent Cytogenomics software (v4.0.1.) was used for evaluation of the CNVs. DNA sequence information refers to the public UCSC database (Assembly: Human GRCh37/hg19). The CNVs detected were compared to known aberrations available in public databases like DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources), the Database of Genomic Variants, Clingen Dosage Sensitivity Map, Clinvar, and Ensembl (among others).