4. Examine Loss of Heterozygosity

We can confirm the validity of the run detection algorithm by examining loss of heterozygosity. In fact, runs of homozygosity (calculated on genotypes) can serve as a proxy for loss of heterozygosity (calculated on B allele frequencies).

The B Allele Frequency spreadsheet in the project represents the B allele frequencies calculated for each marker by the Affymetrix B Allele Frequency Calculation script. This script first combines quantile normalized SNP A and B probe intensities for each marker into a theta value. It then calculates B allele frequencies for each marker. If we locate a run of homozygosity in the heat map containing all ROHs, we can confirm that the algorithm worked correctly by plotting the B allele frequency for the same sample that contained the run.

• Open the Heat Map of Binary ROH Status… and notice the long run on Chromosome 1. For a better view, double click on Chromosome 1 in the Full Domain View to zoom into chromosome 1.
• Click on the long green run to find out which sample it belongs to (it may be easier to zoom into the run to ensure you click on the correct sample). The sample information will appear in the Data Consol window in the bottom left corner. The first row in the data console contains some information about the sample. Make sure you’ve selected sample NA12874.

Now we can add a plot of the B allele frequencies for this sample.

• Click on User Graphs in the Graph Control Interface to bring up the Add Graph tab below it. Change the spreadsheet by clicking on the arrow in the drop down menu after From: and choose Select Spreadsheet. Scroll down to the B Allele Frequencies – Transposed – Sheet 1 and select it, then click OK. This will bring up the samples that you can add to the graph.
• In the Search: box, enter the sample number you identified earlier (NA12874). Check the appropriate sample and click Add.

Figure 18. Heat Map with added B Allele Frequencies

If you zoomed in to select the sample, zoom back out to chromosome one by double clicking on it in the Full Domain View. Your plot should look like Figure 18.

B Allele frequencies (y-axis) close to 1 or 0 correspond to homozygous SNPs whereas a frequency closer to .5 suggests heterozygous SNPs. Notice over the same section as the long run from the heat map (essentially the whole q arm of Chromosome 1), the B allele frequency plot contains relatively few markers in the middle. Alternatively, where there are no runs of homozygosity for this given sample (p arm of Chromosome 1), the data are reasonably distributed between 0, .5 and 1.