Chapter 2 - University of British Columbia
Chapter 2 - University of British Columbia
Chapter 2 - University of British Columbia
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4.2.3 Determining frequent regions <strong>of</strong> UPD, gain and loss<br />
To determine frequent regions <strong>of</strong> gain, loss, and UPD, the frequency <strong>of</strong> each alteration was<br />
determined for each SNP probe on the somatic chromosomes. A frequency threshold <strong>of</strong> 40%<br />
was used. To smooth out regions <strong>of</strong> UPD (and for gain and loss as well), a three step process<br />
was performed. First, adjacent probes with frequencies greater than the threshold were merged<br />
together. Second, to account for dips in frequency where one region is split into two, if the dip is<br />
less than 1 Mb in size, the regions were merged. Finally, smoothed regions <strong>of</strong> UPD, gain, or<br />
loss which were less than 100 probes in size were removed.<br />
4.2.4 Determination <strong>of</strong> UPD in cancer cell lines<br />
Raw SNP 6.0 data (.CEL files) from cancer cell lines were obtained from the Wellcome Trust<br />
Sanger CGP database. CEL files were then genotyped similarly as above to generate CHP<br />
files. To define a total copy number and allele specific copy number reference, SNP 6.0 data,<br />
generated from 72 CEPH HapMap samples, were obtained from Affymetrix and were also<br />
genotyped. Unpaired copy number and allele specific copy number analyses were performed,<br />
as described above, to determine regions <strong>of</strong> allelic imbalance without a change in total copy<br />
number using Partek Genomics Suite.<br />
4.2.5 Expression analysis <strong>of</strong> genes in focal regions <strong>of</strong> UPD<br />
For 16 <strong>of</strong> the 46 tumor/non-malignant tissue pairs, gene expression pr<strong>of</strong>iles on a custom<br />
Affymetrix chip were generated. The 32 samples were normalized using the RMA algorithm<br />
[21] in the Bioconductor s<strong>of</strong>tware suite in R [22]. To determine overexpression in a given<br />
sample pair for a given gene, since expression values are in log2 space, expression values in<br />
non-malignant samples were subtracted from expression values in the tumor. A two-fold<br />
expression change was deemed significant for this analysis.<br />
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