• iKarios
  • NPHL

Learn more about the elements of our methodology

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

250K Nsp Assay

DNA extraction
RE digest
Ligation
PCR x 4
Bioanalyzer x2
Fragmentation
Labeling
Hybridization
Array with 250,000 probe sets

Assay Overview

Genotyping and Copy Number Analysis

Following hybridization and scanning, a data file is created which contains the raw hybridization signal intensities for each probe on the array. The data file is fed into a genotyping algorithm which generates the genotype of every SNP on the array. These files are then used by the CNAGv3.0 software program (Yamamoto G, et al.Am J Hum Genet. 2007 Jul;81(1):114-26.) to obtain log2ratio plots with superimposed loss-of-heterozygosity (LOH) status. The software is linked to the UCSC Genome Browser.

Tumor: Chronic Lypmhocytic Leukemia (CLL)

SNP array Virtual Karyotype of colorectal carcinoma

This SNP array karyotype from a colorectal carcinoma demonstrates allele-specific analysis (red and green plot) generated using the AsCNAR algorithm (Yamamoto G, et al. Am J Hum Genet. 2007 Jul;81(1):114-26). The allele-specific plot provides the relative copy number of each allele separately. In a normal diploid state, there is one copy of the red allele and one copy of the green allele (one from each parent), 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 are 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 are 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.

Note: The tumor-initiating event in this case was copy neutral LOH of the APC gene on chromosome 5q. In otherwords, the 2nd hit for this patient was via acquired uniparental disomy rather than deletion.

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 or Affymetrix GTC software.