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SubtitleText Adding a contrast of Down Syndrome and Normal samples AnchorName Configuring ANOVA Contrasts

 Because the data is log₂ transformed, PGS will automatically detect this and will automatically select Yes in the Data is already log transformed? at the top right-hand corner. PGS will use the geometric mean of the samples in each group to calculate the fold change and mean ratio for the contrast between the Down syndrome and Normal samples.

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SubtitleText ANOVA configuration with contrasts included AnchorName ANOVA Configuration with Contrasts

 

• By default, the Specify Output File is checked and gives a name to the output file. If you are trying to determine which factors should be included in the model and you do not wish to save the output file, simply uncheck this box
• Select OK in the ANOVA dialog to compute the 3-way mixed-model ANOVA
• Several progress messages will display in the lower left-hand side of the ANOVA dialog while the results are being calculated.

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SubtitleText Sources of Variation tab showing a bar chart AnchorName ANOVA Sources of Variation

 This plot presents the mean signal-to-noise ratio of all the genes on the microarray. All the non-random factors in the ANOVA model are listed on the X-axis (including error). The Y-axis represents the mean of the ratios of mean square of all the genes to the mean square error of all the genes. Mean square is ANOVA’s measure of variance. Compare each signal bar to the error bar; if a factor bar is higher than the error bar, that factor contributed significant variation to the data across all the variables. Notice, that this plot is very consistent with the results in the PCA scatter plot. In this data, on average, Tissue is the largest source of variation.

To view the source of variation for each individual gene, right click on a row header in the ANOVA-3way (ANOVAResults) spreadsheet and select the Sources of Variation item from the pop-up menu. This generates a Sources of Variation tab for the individual gene. View a few Sources of Variation plots from rows at the top of the ANOVA table and a few from the bottom of the table.

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SubtitleText Calling an ANOVA Interaction Plot for a gene AnchorName Calling ANOVA Interaction Plot

 Generate these plots for rows 3 (DSCR3) and 8 (CSTB). If the lines in this plot are not parallel, then there is a chance there is an interaction between Tissue and Type. DSCR3 is a good example of this (Figure 7). We can look at the p-values in column 9, p-value(Type * Tissue) to check if this apparent interaction is statistically significant. 

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SubtitleText Creating a gene list from ANOVA results AnchorName List Manager

 

The spreadsheet Down_Syndrome_vs_Normal (A) will be created as a child spreadsheet under the Down_Syndrome-GE spreadsheet.

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SubtitleText Generating a Volcano Plot from ANOVA results AnchorName Generating Volcano Plot

 

• Set X Axis (Fold-Change) to 12. Fold-Change(Down Syndrome vs. Normal), and the Y axis (p-value) to be 110. p-value(Down Syndrome vs. Normal)
• Select OK to generate a Volcano Plot tab and for genes in the ANOVA spreadsheet (Figure 10)
  
  

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SubtitleText Volcano plot generated from ANOVA spreadsheet AnchorName Volcano Plot

 In the plot, each dot represents a gene. The X-axis represents the fold change of the contrast, and the Y-axis represents the range of p-values. The genes with increased expression in Down syndrome samples on the right side; genes with reduced expression in Down syndrome samples are on the left of the N/C line. The genes become more statistically significant with increasing Y-axis position. The genes that have larger and more significant changes between the Down syndrome and normal groups are on the upper right and upper left corner. 

In order to select the genes by fold-change and p-value, we will draw a horizontal line to represent the p-value 0.05 and two vertical lines indicating the –1.3 and 1.3-fold changes (cutoff lines).

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SubtitleText Setting cutoff lines for -1.3 to 1.3 fold changes and a p-value of 0.05 AnchorName Set Cutoff Lines

 

• Select OK to draw the cutoff lines
• Select OK in the Plot Rendering Properties dialog to close the dialog and view the plot

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SubtitleText Creating a gene list from a volcano plot AnchorName Create List from Volcano Plot

 Note: If no column is selected in the parent (ANOVA) spreadsheet, all of the columns will be included in the gene list; if some columns are selected, only the selected columns will be included in the list.

• Specify a name for the gene list (example: volcano plot list) and write a brief description about the list. The description is shown when you right-click on the spreadsheet > Info > Comments. Here, I have named the list "volcano plot list" and described it as "Genes with >1.3 fold change and p-value <0.05" (Figure 13). 

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