EXPERIMENTAL OVERVIEW

1. Comparative genomic hybridization (CGH) is a technique for studying chromosomal changes in cancer. As cancerous cells multiply, they can undergo dramatic chromosomal changes, including chromosome loss, duplication, and the translocation of DNA from one chromosome to another. Chromosome aberrations have previously been detected using optical imaging of whole chromosomes, a technique with limited sensitivity, resolution, quantification, and throughput. Efforts in recent years to use microarrays to overcome these limitations have been hampered by inadequate sensitivity, specificity and flexibility of the microarray systems. The Agilent oligonucleotide CGH microarray system overcomes several scientific hurdles that have impeded comparative genomic studies of cancer. This new system can reliably detect single copy deletions in chromosomes. The system includes a whole human genome microarray, reagents for sample preparation, an optimized microarray processing protocol, and software for data analysis and visualization. In this study, we demonstrate the ability of the Agilent oligo 244K CGH system to reliably detect DNA copy variations with reduced sample input amounts. Further, we illustrate the benefits of high resolution CGH in mapping chromosomal breakpoints in cancer cells. EXPERIMENTAL OVERVIEW • The Human Genome CGH 244K microarray (p/n G4411B) and the Human Genome CGH 44K (p/n G4410B) were used, which contained In situ synthesized 60 mer oligo probes covering both the coding and non-coding sequences of total genomic DNA with an average spatial resolution of approximately 6 kb and 35 kb, respectively. • Total genomic DNA was used for direct labeling. Human genomic DNA from pooled Male, Female, and colon carcinoma cell line HT-29 were labeled using the Agilent Genomic DNA Labeling Kit PLUS (p/n 5188-5309). Each reaction started with 0.2 mg, 0.5 mg, or 3 mg of total genomic DNA input, and the entire reaction product was hybridized onto the microarray using the Agilent aCGH Hybridization Kit (p/n 5188-5220) using Agilent standard CGH protocol (version 4.0). The data in each graph represents an average of two or more microarrays. • Microarrays were washed using the Agilent aCGH Wash Kit (p/n 5188-5226), and scanned using the Agilent dual laser DNA microarray scanner (p/n G2565BA). Data were extracted using Agilent’s Feature Extraction version 9.1 software. Data were analyzed using Agilent’s CGH Analytics version 3.4 software. Quantitative analysis to assess the performance of the complete Agilent oligo aCGH microarray system. Sunny Song, Peter Webb, Anniek De Witte, Diane Ilsley and Jim Collins Agilent Technologies, Santa Clara, CA 95051 ABSTRACT Fig. 4 244K microarray platform performance using a range of genomic DNA input masses. A) Log Ratio Noise.The standard deviation of Log2 Ratio values is < 0.25 across the range of input masses tested.B) Reproducibility across replicate probes.The %CV values for intra-array replicate probes are all less than < 5 % for the different DNA input masses. C) Accuracy.The average Log2 Ratio of chrX probes in Male/Female hybridizations is greater than 0.9 across the DNA input masses tested indicating a high level of accuracy. Fig. 5 The high resolution 244K microarray provides enhanced precision that enables novel aberration detection. CGH Analytics views of Agilent 244K and 44K analyses of chromosome 3 (A, B) and chromosome 16 (C, D) in the human colon carcinoma cell line HT29. A)Left, Scatter plot (chromosome view) produced from Human CGH Microarray 244K analysis reveals multiple 3p arm deletions in regions such as 3p12.1-12.3 (horizontal shift to left of zero line) and a focal deletion in 3p14.2 (horizontal shift to left of zero line, outlined by dotted blue box). Right, Zoomed-in gene view which focuses on a 7 MB window within 3p14.2 containing the focal deletion. It distinctly shows two different deletion patterns (homozygous and heterozygous deletion) both within the single tumor suppressor FHIT gene.B) Parallel scatter plots (chromosome and gene views, respectively) from CGH 44K Microarray analysis of the same sample as in panel A. C)Left, Scatter plot (chromosome view) produced from 244K analysis reveals a focal homozygous deletion in 16p13.2. Right, Zoomed-in gene view which focuses on a 0.5 MB window within 16p13.2 containing a homozygous deletion. It readily detects multiple deletion patterns within the ataxin-2 binding protein A2BP1 gene (green dots). Fig. 2. Probe coverage on the 244K microarray.Typical probe coverage characteristics on the Human Genome CGH 244K and 44K microarrays illustrated using the UCSC hg18 Human Genome Browser, Feb 2006 (NCBI build 36) of human chromosome 17. At the top of each view, each short vertical bar represents a single probe on Agilent Human Genome CGH 244K (red) or 44K (blue) microarray. Annotations below the probes indicate the location mapping of UCSC known genes (blue or black) and Sanger microRNAs (red). A) Variable probe density. A 3.0 MB microarray window of 17q24-25 shows comprehensive coverage that is more dense in coding regions. B) Coverage of long genes. A 3.0 MB window of 17q11-12 depicts evenly distributed probe coverage across long (ACCN1) as well as short (NLE1) genes. C) Coverage of miRNAs. A 1.0 MB region of 17q11.2 illustrates microRNA representation (hsa-mir-193, hsa-mir-365-2). Further, it shows a ~ 50KB microdeletion within 16q23.1 (green dots in red circle). This microdeletion (heterozygous deletion) was verified by multiple consecutive probes.D) Parallel scatter plots from CGH 44K Microarray analysis of the same sample as in panel C. Fig. 3.Probe Performance on the 244K microarray.A) Low noise and high specificity. aCGH 244K microarray log2 ratios are shown for male/female hybridizations.A representative separation histogram showing the distribution of red/green fluorescence ratios for the autosomal probes (green) and X-chromosomal probes (blue), plotted as fraction of probes on the ordinate (female, XX) versus red/green fluorescence ratio (binned by intervals) on the abscissa of the X-chromosome (male, XY) on the Agilent Human Genome CGH 244K microarray. B) Improved signal intensity.Signal intensities of probes on the 244K microarray show a tighterdistribution and medians of log10 signal Intensities. 244K microarray (red) and 44K microarray (blue) were hybridized with male/female DNA. More probes on the 244K microarray show higher signal intensities while the distribution of these intensities is tighter. Fig. 1. Agilent’s Microarray Comparative Genomic Hybridization (CGH) Whole Platform Solution. Schematic depiction of the procedures used to measure DNA copy-number changes by CGH microarray hybridization. Genomic DNA samples isolated from tumor cells and normal cells are labeled with two different fluorophores (Cy5 and Cy3, respectively) and hybridized together to a single CGH microarray. For each DNA probe on the microarray, the ratio of intensity of the fluorescence measured for the two fluors is determined by dual laser scanning and data are analyzed using the CGH Analytics software. A red color represents increased DNA copy number, green represents decreased copy number (i.e., deletion), and yellow represents no change in DNA copy number in tumor cell DNA compared with normal cell DNA. • CONCLUSIONS • Agilent’s 244K CGH microarray platform allows researchers to identify DNA copy variations with high resolution on a genome-wide scale. • High quality data can be obtained from low input masses of genomic DNA without the need for sample amplification or complexity reduction. • This high density platform provides enhanced precision that enables novel aberration detection.