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SubtitleText ANOVA spreadsheet AnchorName ANOVA Spreadsheet

For additional information about ANOVA in PGS, see Chapter 11 Inferential Statistics in the User’s Manual (Help > User’s Manual).

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Visualizing ANOVA Results

Deciding which factors to include in the ANOVA may be an iterative process while you decide which factors and interactions are relevant as not all factors have to be included in the model. For example, in this tutorial, Gender and Scan date were not included.  The Sources of Variation plot is a way to quantify the relative contribution of each factor in the model towards explaining the variability of the data.

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SubtitleText Interaction Plot for DSCR3 AnchorName Interaction Plot

We can view the expression levels for a gene for each sample using a Dot Plot.

• Right click on the gene row header and select Dot Plot (Orig. Data) from the pop-up menu. This generates a Dot Plot tab for the selected gene (Figure 8)
  
  

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SubtitleText Dot Plot showing DSCR3 expression levels for each sample AnchorName Dot Plot
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In the plot, each dot is a sample of the original data. The Y-axis represents the log₂ normalized intensity of the gene and the X-axis represents the different types of samples. The median expression of each group is different from each other in this example. The median of the Down syndrome samples is ~6.3, but the median of the normal samples is ~6.0. The line inside the Box & Whiskers represents the median of the samples in a group. Placing the mouse cursor over a Box & Whiskers plot will show its median and range. 

Create Gene List

Now that you have obtained statistical results from the microarray experiment, you can now take the result of 22,283 genes and create a new spreadsheet of just those genes that pass certain criteria. This will streamline data management by focusing on just those genes with the most significant differential expression or substantial fold change. In PGS, the List Manager can be used to specify numerous conditions to use in the generation of our list of genes of interest. In this tutorial, we are going to create a gene list with a fold change between -1.3 to 1.3 with an unadjusted p-value of < 0.0005. 

• Invoke the List Manager dialog by selecting Create Gene List in the Analysis section of the Gene Expression workflow
• Ensure that the 1/ANOVA-3way (ANOVAResults) spreadsheet is selected as this is the spreadsheet we will be using to create our new gene list as shown (Figure 89)
• Select the ANOVA Streamlined tab. In the Contrast: find genes that change between two categories panel, choose Down Syndrome vs. Normal and select Have Any Change from the Setting dropdown menu list. This will find genes that have a fold change different between the types of samples
• In the Configuration for “Down Syndrome vs Normal” panel, check that Include size of the change is selected and enter 1.3 into Fold change >  and -1.3 in OR Fold change <
• Select Include significance of the change, choose unadjusted p-value from the dropdown menu, and < 0.0005 for the cutoff. The number of genes that pass your cutoff criteria will be shown next to the # Pass field. In this example, 23 genes pass the criteria. 
• Set Save the list as A, select Create, and then select Close to view the new gene list spreadsheet
  
  

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• Select the 1/ANOVA-3way (ANOVAResults) spreadsheet in the Analysis tab. Thisis the spreadsheet we will be using to create the gene list
• Select View > Volcano Plot from the PGS main menu (Figure 910)
  
  

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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 1011)
  
  

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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. 

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• Select Rendering Properties ()
• Choose the Axes tab
• Check Select all points in a section to allow PGS to automatically select all the points in any given section
• Select the Set Cutoff Lines button and configure the Set Cutoff Lines dialog as shown (Figure 1112)
  
  

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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

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• Right-click on the selected region in the plot and choose Create List to create a list including the genes from the section selected (Figure 1213). Note that these p-values are uncorrected
  
  

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• 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 1314). 

 The list can be saved as a text file (File > Save As Text File) for use in reports or by downstream analysis software.

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