Agilent oligonucleotide array based cgh for genomic dna analysis

DNA processing, microarray handling and data analysis were performed according to the protocol by manufacturer (Agilent Oligonucleotide Array‐Based CGH for Genomic DNA Analysis, version 6.1, August 2009, Agilent Technologies, CA, USA) with minor modifications. Oligonucleotide‐based Human Genome Microarrays (Agilent Technologies) containing 60 K, 180 K, 400 K and 1 M features were used for hybridization, among which 2 × 400 K format was used to examine genome‐wide copy number alteration (Figure S1). The 4 × 180 K format targeting amplified regions was customized microarray that we created by using the Agilent eArray online system (https://earray.chem.agilent.com/) for interrogating the genome of DMs with high‐resolution and accuracy. Data were quality controlled and extracted using Feature Extraction (version 9.1, Agilent Technologies, CA, USA), and subsequently analysed by Agilent Genomic Workbench (version 7.0, Agilent Technologies, CA, USA).

Genomic DNA was quantified by spectrophotometry (NanoDrop ND1000, NanoDrop Technologies, Wilmington, Delaware USA). Integrity of DNA was assessed by 0.8% agarose gel electrophoresis. Non-amplification labeling of DNA (direct method) was obtained following the ‘Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis’ protocol Version 4.0 (Agilent Technologies, Palo Alto, California USA. p/n G4410-90010). 500 ng of experimental and pool female reference genomic DNA samples were fragmented in a restriction digestion step. Digestion was confirmed and evaluated by DNA 7500 Bioanalyzer assay. Cyanine 3-dUTP and cyanine 5-dUTP were used for fluorescent labeling of test and reference digested gDNAs respectively, using the ‘Agilent Genomic DNA Labeling Kit PLUS’ (Agilent p/n 5188-5309) according to the manufacturer's instructions. Labeled DNA was hybridized with Human Genome CGH Microarray 44K (Agilent p/n G4426B-014950) containing 43,000+ coding and noncoding human sequences. Arrays were scanned in an Agilent Microarray Scanner (Agilent G2565BA) according to the manufacturer's protocol and data extracted using Agilent Feature Extraction Software 9.5.3.1 following the Agilent protocol CGH-v4_95_Feb07 (‘Lowess Only’ normalization correction dye bias method instead of ‘Linear Only’) and the QC Metric Set CGH_QCMT_Feb08.