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November 01, 2010 (Vol. 30, No. 19)

Improving LOH Detection on CGH Microarrays

Technology Strives to Bypass a Number of Limitations of Current Products

  • Protocol

    The sample-preparation and hybridization protocol is exactly the same as that for CGH-only microarrays. We digested genomic DNA from an unknown test sample and a control sample with known genotype with AluI and RsaI. Then we labeled the test sample with Cy5 dye and the control sample with Cy3 dye and hybridized both samples to the same array. After washing, we scanned the slides at 3 micron resolution on Agilent’s High Resolution C Scanner and extracted and analyzed the images using Agilent Genomic Workbench 6.5 software.

  • Methodology

    Click Image To Enlarge +
    Figure 1. Schematic for the Agilent CGH+SNP microarray workflow: Three possible cases for one SNP example are shown: homozygous CC, heterozygous CA, and homozygous AA. For each case, homologous regions of two chromosomes are shown, with the two possible alleles colored in red and/or blue. (A) Neither of the SNP sites is cut by AluI or RsaI, which yields the highest signal level on the microarray. (B) One of the SNP sites is cut by AluI or RsaI, which yields an intermediate signal level. (C) Both of the SNP sites are cut by AluI or RsaI, which yields the lowest signal level.

    Our method is similar to restriction fragment length polymorphism (RFLP). In RFLP analysis a genomic DNA sample is digested by restriction enzymes and the resulting fragments are separated by gel electrophoresis. Instead of using gel electrophoresis we use a microarray.

    Restriction enzymes cut molecules of DNA at specific recognition sites, so cutting with a particular enzyme should always produce the same size and number of fragments. However, when a site is polymorphic, or existing as two alleles, the restriction enzyme will not recognize one allele and will not cut there, leading to a length polymorphism.

    The ~60,000 SNP probes on the CGH+SNP microarray span variant AluI or RsaI restriction enzyme recognition sites and measure the copy number of the uncut allele at those loci (Figure 1). We measure the total copy number of the region encompassing the SNP site by neighboring CGH probes. The copy number of the cut allele can then be inferred from the total copy number and the copy number of the uncut allele.

    A SNP copy-number call is made from the log ratio of the test sample (Cy5 signal) versus a genotyped internal reference (Cy3 signal). The copy number of each SNP in the reference sample is known. In order to determine the allele-specific copy number of the test sample, the log ratios of the SNP probes are adjusted by the copy number of the reference sample.

    The reference-adjusted log ratios fall into three categories corresponding to the copy numbers of the uncut alleles in the sample, which correspond to the three possible diploid genotypes for the SNPs: AA, AB, or BB. Regions of copy-neutral LOH or UPD are then located by identifying genomic regions with a statistically significant scarcity of heterozygous SNP calls.

  • Results/Conclusions

    Click Image To Enlarge +
    Figure 2. Agilent Genomic Workbench view of SNP data (number of uncut alleles, bottom panel), and CGH data (log2 ratios, top panel) from a CGH+SNP array shows UPD of the entire chromosome 15. Settings for CGH aberration calling: ADM-2, threshold 5, minimum of 3 probes Ž0.25 log2 ratio.

    With high-quality DNA samples, the SNP call rate is greater than 95% with greater than 99% accuracy, and the presence of SNP probes does not affect the performance of CGH probes. The number and quality of copy number aberrations detected on the SurePrint G3 CGH+SNP microarrays is comparable to detection using SurePrint G3 CGH-only microarrays with the added benefit of simultaneous identification of copy-neutral aberrations.

    For a normal diploid region of the genome, one expects the 0, 1, and 2 SNP copy numbers of the uncut allele to be randomly distributed. In a diploid genome carrying a copy-neutral LOH or UPD aberration, the SNP probes will only report alleles that are homozygously cut and uncut (0 and 2 uncut alleles) and, therefore, only two states are found. Figure 2 shows an example of UPD observed in an individual known to have genomic aberrations associated with Angelman syndrome.

    In conclusion, the SurePrint G3 CGH+SNP microarray affords the precision of SNP detection to measure copy-neutral genomic changes with ~5–10 Mb resolution as well as the reproducibility of Agilent’s CGH platform—all on a single microarray. As a result, researchers no longer need to choose between high-resolution, high-quality CGH data and the detection of LOH/UPD or alternatively run two separate experiments.

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