For more information about Virtual Karyotypes go to the Wikipedia site for Virtual Karyotype that was initiated by Dr. Hagenkord.

How Do We Generate a Virtual Karyotype?
1. Enrich for clonal population, if necessary.
2. Extract genomic DNA.
3. GeneChip Mapping Assay.
4. Genotyping and Copy Number Analysis.
5. Karyogram and reporting.

Mapping Assay Overview

Genotyping and Copy Number Analysis
Following hybridization and scanning, a .CEL file is created which contains the raw hybridization signal intensities for each probe on the array. The .CEL file is fed into a genotyping algorithm, Affymetrix Genotyping Console (GTC), which generates a .CHP containing the genotype of every SNP on the array. The .CEL file and .CHP file are then used by the CNAGv3.0 software program (Yamamoto G, Nannya Y, Kato M, Sanada M, Levine RL, Kawamata N, et al. Highly sensitive method for genomewide detection of allelic composition in nonpaired, primary tumor specimens by use of affymetrix single-nucleotide-polymorphism genotyping microarrays. Am J Hum Genet. 2007 Jul;81(1):114-26.) to obtain log2ratio plots with superimposed loss-of-heterozygosity (LOH) status. The software enables the medical professional signing out the case to zoom in and zoom out of the genome as necessary, and it is linked to the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway) for real-time genome surfing.

The copy number plot has been linearized for ease of interpretation. Typically, copy number plots from virtual karyotypes are plotted as a log2ratio of the hybridization intensity signal of the tumor/normal for each SNP on the array. Don't let this confuse you too much. Just think of the zero line as diploid (like I have it annotated here). When the line pops up, that is a gain of genetic material. When it pops down from zero, that is a loss. Each SNP on the array has its log2ratio plotting in chromosomal order from right to left along the x-axis. In addition, the CNAG software color-codes each chromosomes. The green heterozygous call bars are placed by the software every time it detects and AB call in the tumor. If there is an extended run where there are no AB calls in the tumor (ie, a loss of heterozgygosity), then the Hidden Markov Model (HMM) for LOH likelihood will trigger (yellow = low LOH likelihood, blue = high LOH likelihood). There is also a HMM for copy number that assessing what the neighboring SNPs are doing, bins them into a whole number, and color codes them: dark blue = 0, light blue = 1, yellow = normal/diploid, pink = 3, pink-red = 4, red-pink = 5, and bright red = greater than 5 (amplification).

In this plot, I have added an allele-specific analysis plot (red and green) generated using the AsCNAR algorithm (Yamamoto G, Nannya Y, Kato M, Sanada M, Levine RL, Kawamata N, et al. Highly sensitive method for genomewide detection of allelic composition in nonpaired, primary tumor specimens by use of affymetrix single-nucleotide-polymorphism genotyping microarrays. Am J Hum Genet. 2007 Jul;81(1):114-26). It is a little confusing at first, but highly informative once you get used to it. The allele-specific plot tells you the relative dose of each allele at each SNP, and it can only be obtained from SNP-based arrays. In a normal diploid state, there is one copy of the red allele and one copy of the green allele, for an overall copy number of two (e.g., chromosome 2 in this tumor). When there is copy neutral LOH/acquired UPD, both copies are coming from the same parent, so there is two copies of the red allele and zero copies of the green allele, for an overall copy number of two (e.g., 5q in this tumor). In a trisomy, there is two copies of the red allele and one copy of the green allele, for an overall copy number of 3 (e.g., chromosome 7 in this tumor). A deletion is shown for 11q in this tumor. There is one copy of the red allele and zero copies of the green allele for an overall copy number of 1.

Karyogram and Report Generation
Using the data files generated by CNAGv3.0, OneClickCGH (InfoQuant, LTD, London, UK) is used for breakpoint determination, ISCN nomenclature conversion, karyogram, and report generation. Go to Sample Report.