Investigation Title Comparative genomic hybridization of samples from 30 humans and 30 chimpanzees to survey copy number variation in these species Comment[Submitted Name] Comparative survey of copy number variation in chimpanzees and humans Experimental Design comparative_genome_hybridization_design strain_or_line_design species_design comparative genomic hybridization by array Experimental Design Term Source REF mo mo mo EFO Comment[ArrayExpressReleaseDate] 2008-08-28 Comment[SecondaryAccession] Comment[AEMIAMESCORE] 4 Comment[ArrayExpressAccession] E-TABM-479 Comment[MAGETAB TimeStamp_Version] 2010-10-16 13:06:59 Last Changed Rev: 14677 Experimental Factor Name Individual Organism Experimental Factor Type individual organism Experimental Factor Term Source REF Person Last Name Clayton Person First Name Stephen Person Mid Initials Person Email sc13@sanger.ac.uk Person Phone Person Fax Person Address Person Affiliation Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2008-08-28 PubMed ID 18775914 Publication DOI 18775914 Publication Author List Perry, George H.; Yang, Fengtang; Marques-Bonet, Tomas; Murphy, Carly; Fitzgerald, Tomas; Lee, Arthur S.; Hyland, Courtney; Stone, Anne C.; Hurles, Matthew E.; Tyler-Smith, Chris; Eichler, Evan E.; Carter, Nigel P.; Lee, Charles; Redon, Richard Publication Title Copy number variation and evolution in humans and chimpanzees Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Sixty-one array-CGH experiments were performed on the human WGTP platform, comparing: (1) 30 unrelated chimpanzees to a single chimpanzee reference individual, (2) 30 unrelated humans to a single human reference individual and (3) the chimpanzee reference individual to the human reference individual. Protocol Name P-TABM-2644 P-TABM-2643 P-TABM-510 P-TABM-508 P-TABM-512 P-TABM-2642 Protocol Type grow nucleic_acid_extraction labeling hybridization image_acquisition bioassay_data_transformation Protocol Description Human DNAs were obtained from the Coriell Institute for Medical Research and the HGDP-CEPH Human Genome Diversity Cell Line Panel. Chimpanzee B-lymphoblast cell lines were obtained from the Coriell Institute for Medical Research and Integrated Primate Biomaterials and Information Resource. Chimpanzee whole blood samples were collected at the New Iberia Research Center and the Primate Foundation of Arizona during routine veterinary appointments. DNA was isolated from cell lines and whole blood using the PureGene DNA Isolation Kit (Gentra Systems). Test and reference DNA samples were differentially labeled using the Bioprime labeling kit (Invitrogen Carlsberg, CA, USA) with modifications of the nucleotide mix. Briefly, a 260 ?l reaction was set up containing 300 ng of DNA and 120 ?l of 2.5x random primer solution. After denaturing the DNA for 10 minutes at 100°C, 30 ?l of 10x dNTP mix (1 mM dCTP, 2 mM dATP, 2 mM dGTP, and 2 mM dTTP in TE buffer), 3 ?l of 1 mM Cy5-dCTP or Cy3-dCTP (NEN Life Science Products), and 6 ?l of Klenow fragment were added on ice to a final reaction volume of 300 ?l. The reaction was incubated at 37°C overnight and stopped by adding 30 ?l of stop buffer supplied in the kit. Unincorporated nucleotides were removed by use of Microcon YM-300 Filter Devices (Millipore Co.), according to the suppliers’ instructions. Hybridizations were carried out on a Tecan HS TM Hybridization Station (Tecan Group Ltd.) using 63x20 mm chambers. Cy3 and Cy5 labeled DNAs were combined, precipitated with 270 ?g of human Cot1 DNA (Roche Diagnostics Ltd., UK) and resuspended in 165 ?l of hybridization buffer (50% formamide, 5% dextran sulfate, 0.1% Tween 20, 2x SSC and 10 mM Tris/HCl, pH 7.4, 10 mM Cysteamine). Prehybridization solution was prepared simultaneously by precipitating 100 ?l of herring sperm DNA (10 mg/ml, Sigma Aldrich, UK) and resuspending in 165 ?l of hybridization buffer. The prehybridization and hybridization solutions were then denatured for 10 min at 72°C. The prehybridization solution was injected into the Tecan chamber following instructions displayed on the station. During prehybridization (45 min at 37 oC), the hybridization solution was incubated at 37°C. Hybridization was carried out for 21 hours at 37oC with medium agitation frequency. Slides were washed with PBS/Tween 20/2mM cysteamine (wash time 0.5 min, soak time 0.5 min, 15 Cycles at 37oC), 0.1 x SSC (wash time 1.0 min, soak time 2.0 min, 5 Cycles at 54oC), PBS/Tween 20/2mM cysteamine (wash time 0.5 min, soak time 0.5 min, 10 Cycles at 23oC) and HPLC water (wash time 0.5, soak time 0.0, 1 Cycle at 23oC) before drying for 2.5 min using nitrogen gas. All experiments were performed in duplicate with DNA labeling color reversal (dye swap). Array images were acquired using an Agilent laser scanner (Agilent Technologies, UK). After block normalisation, data analysis was performed using custom Perl scripts. Log2 ratios were normalized by linear regression against clone GC percent using the module �'LineFit'� (http://search.cpan.org/~randerson/Statistics-LineFit-0.07) and then median-normalised chromosome by chromosome. The ratios for each clone in the two dye swap hybridizations were then averaged, only if they differed by less than 50% (i.e. less than a difference of 0.585 on the log2 scale). The 68.2th percentile of the absolute values for all combined ratios was then calculated chromosome by chromosome as an estimation of the standard deviation (SDe). Clones reporting replicates different by more than eight times the SDe were excluded from further analysis. Dye-swap experiments were accepted for CNV calling only if the following criteria were fulfilled: (1) Global SDe < 0.06; (2) Global clone exclusion rate < 10%; (3) Clone exclusion rate per individual chromosome < 20%. Clones contained in chromosomes with a corresponding chromosomal median of > 0.1 or < -0.1 were flagged and excluded from CNV calling. Thus, chromosomes X and Y in female versus male results, as well as chromosomal artefacts (aneusomies or very large imbalances) were automatically excluded from calling. Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology SDRF File E-TABM-479.sdrf.txt Term Source Name mo The MGED Ontology ArrayExpress mo EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version