Digital karyotyping

Abstract

Alterations in the genetic content of a cell are the underlying cause of many human diseases, including cancers. We have developed a method, called digital karyotyping, that provides quantitative analysis of DNA copy number at high resolution. This approach involves the isolation and enumeration of short sequence tags from specific genomic loci. Analysis of human cancer cells by using this method identified gross chromosomal changes as well as amplifications and deletions, including regions not previously known to be altered. Foreign DNA sequences not present in the normal human genome could also be readily identified. Digital karyotyping provides a broadly applicable means for systematic detection of DNA copy number changes on a genomic scale.

Fig 1.

Schematic of the digital karyotyping approach. Colored boxes represent genomic tags. Small ovals represent linkers. Large blue ovals represent streptavidin-coated magnetic beads. See text for details.

Fig 2.

Low-resolution tag density maps reveal many subchromosomal changes. The upper graph in each set corresponds to the digital karyotype, while the lower graph represents CGH analysis. An ideogram of each normal chromosome is present under each set of graphs. For all graphs, values on the y axis indicate genome copies per haploid genome, and values on the x axis represent positions along the chromosome (Mb for the digital karyotype; chromosome bands for CGH). Digital karyotype values represent exponentially smoothed ratios of DiFi tag densities, using a sliding window of 1,000 virtual tags normalized to the NLB genome. Chromosomal areas lacking digital karyotype values correspond to unsequenced portions of the genome, including heterochromatic regions. Note that using a window of 1,000 virtual tags does not permit accurate identification of alterations less than ≈4 Mb, such as amplifications and homozygous deletions, and smaller windows need to be used to accurately identify these lesions (see Fig. 3 for an example).

Fig 3.

High-resolution tag density maps identify amplifications and deletions. (A) Amplification on chromosome 7. (Top) A bitmap viewer with the region containing the alteration encircled. The bitmap viewer is comprised of ≈39,000 pixels representing tag density values at the chromosomal position of each virtual tag on chromosome 7, determined from sliding windows of 50 virtual tags. Yellow pixels indicate tag densities corresponding to copy numbers of <110, while black pixels correspond to copy numbers ≥110. (Middle) An enlarged view of the region of alteration. (Bottom) A graphical representation of the amplified region with values on the y axis indicating genome copies per haploid genome and values on the x axis representing positions along the chromosome in Mb. (B) Homozygous deletion on chromosome 5. Top, Middle, and Bottom are similar to those for A except that the bitmap viewer for chromosome 5 contains ≈43,000 pixels, tag density values were calculated in sliding windows of 150 virtual tags, and yellow pixels indicate copy numbers >0.1 while black pixels indicate copy numbers ≤0.1. (Bottom) Below the graph is a detailed analysis of the region containing the homozygous deletion in DiFi and Co52. For each sample, white dots indicate markers that were retained, while black dots indicate markers that were homozygously deleted. PCR primers for each marker are listed in Table 4.

Fig 4.

Identification of EBV DNA in NLB cells. NLB, genomic tags derived from NLB cells after the removal of tags matching human genome sequences or tags matching DiFi cells. DiFi, genomic tags derived from DiFi cells after the removal of tags matching human genome sequences, or tags matching NLB cells. The number of observed tags matching EBV, other viral, or bacterial sequences is indicated on the vertical axis.