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• Under the Visualization section in the Gene Expression workflow, select Cluster Based on Significant Genes (Figure 1)
  
  

• The Cluster Significant Genes dialog asks you to specify the type of clustering you want to perform. Select Hierarchical Clustering and select OK
• Choose the Down_Syndrome_vs_Normal (A)  spreadsheet under the Spreadsheet with differentially expressed genes 
• Choose the Standardize – shift genes to mean of zero and scale to standard deviation of one under the Expression normalization panel. This option will adjust all the gene intensities such that the mean is zero and the standard deviation is 1
• Select OK to generate a Hierarchical Clustering tab (Figure 2)
  
  

The graph (Figure 2) illustrates the standardized gene expression level of each gene in each sample. Each gene is represented in one column, and each sample is represented in one row. Genes with no difference in expression have a value of zero and are colored black. Genes with increased expression in Down syndrome samples have positive values and are colored red. Genes with reduced expression in Down syndrome samples have negative values and are colored green. Down syndrome samples are colored red and normal samples are colored orange. On the left-hand side of the graph, we can see that the Down syndrome samples cluster together.

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• In the Down_Syndrome_vs_Normal (A) spreadsheet, right click on the second column header 2. ProbesetID and select Insert Annotation from the pop-up menu (Figure 3)
  
  

• Select Chromosomal Location under the Column Configuration panel (Figure 4). Leave everything else as default and select OK
  
  

Interestingly, of the 23 genes of the Down_Syndrome_vs_Normal (A) spreadsheet, 20 genes are located on chromosome 21. This suggests that the gene expression changes associated with Down syndrome observed in this study are primarily located on chromosome 21, not distributed throughout the genome, an important finding of this study. 

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