Pathway Analysis using Partek Genomics Suite 6.6 and Partek Pathway

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1 Pathway Analysis using Partek Genomics Suite 6.6 and Partek Pathway Overview Partek Pathway provides a visualization tool for pathway enrichment spreadsheets, utilizing KEGG and/or Reactome databases for human, rat and mouse. The screenshots shown below may vary across platforms and across different versions of Partek. Description of the Data Set This guide uses the Affymetrix Gene ST dataset from the Analyzing microrna Data and Integrating microrna with Gene Expression Data in Partek Genomics Suite 6.6 tutorial. Data and associated files for this tutorial can be downloaded by going to Help > On-line Tutorials from the Partek main menu. The data can also be downloaded directly from: Importing files To proceed with the tutorial data, open the Partek pre-imported tutorial data that is already in a Partek project format (.ppj): mirnamrna integration. This.ppj file contains the Partek format file Affy_miR_BrainHeart_intensities.fmt with all the microrna data, and the Partek format file Affy_HuGeneST_BrainHeart_GeneIntensities.fmt with all the mrna data. For the convenience of this tutorial, an ANOVA was performed in the mrna data and is opened as a child spreadsheet under the Affy_HuGeneST_BrainHeart_GeneIntensities.fmt spreadsheet. Select File > Open Project to invoke the project browser and open the mirnamrna integration project - 3 spreadsheets will now be available: 1. Affy_miR_BrainHeart_intensities 2. Affy_HuGeneST_BrainHeart_GeneIntensities 3. ANOVAResults gene (this is a child spreadsheet of the HuGeneST Sheet) Partek Pathway Quick Start Guide 1

2 Streamlined ANOVA The ANOVA spreadsheet contained within this dataset has rows; before performing pathway analysis this list will be filtered to give a list of significant regulated genes between the two organs. Invoke the Gene Expression workflow Highlight the ANOVA spreadsheet Select Create Gene List from the Analysis section as you have already highlighted an ANOVA spreadsheet you now have the ANOVA Streamlined tab selected automatically Configure as shown (Figure 1) and click Create a new list of 420 genes will be created Figure 1: Configuring the ANOVA Streamlined Close the List Manager Select the newly created list (it will be a child of the HuGeneST file) Select Pathway Analysis from the Biological Interpretation section Use the default settings (Figure 2) Partek Pathway Quick Start Guide 2

3 Figure 2: Pathway Analysis Click Next Again, use the default settings (Figure 3) Figure 3: Pathway Enrichment Click Next Select the database required from the drop down menu (circled red in Figure 4 below) KEGG and Reactome are available Select the species required (human in this example) Ensure the column containing the Gene Symbol is correct Optionally you can include the ANOVA table to parse in Pathway. This enables you to additionally visualize fold-changes or p-values for all genes Partek Pathway Quick Start Guide 3

4 inside the pathways which are targeted by the array you used, even if they are not significant (and therefore not in your selection). Figure 4: Species If you use Pathway on the selected species for the first time, Pathway will automatically download the Kegg information needed for the analysis. Once the pathway enrichment calculation has been performed a new spreadsheet is created (Pathway-Enrichment.txt) and Partek Pathway will be launched (Figure 5) Figure 5: Partek Pathway Partek Pathway Quick Start Guide 4

5 The default pathway to be visualized is that with the highest enrichment score for confirmation, examine the pathway in row 1 in GS (Figure 6) to confirm GABAergic synapse as the most enriched pathway: Figure 6: Pathway Enrichment Spreadsheet Note that this sheet is also included in Pathway. For each Pathway in the Enrichment spreadsheet it is now possible to perform several actions. Right-click on the row header to invoke the contextual menu (Figure 7): Figure 7: Pathway options Export genes in Pathway will create a new spreadsheet in Genomics Suite as a child of the Enrichment spreadsheet it contains all of the genes from the selected pathway. Export genes in list and in pathway creates a smaller list of only the genes from your list that are present in the selected pathway. Show Pathway opens the selected pathway in Partek Pathway. Partek Pathway Quick Start Guide 5

6 Description of Score columns in the Enrichment sheet: Enrichment Score Tissue/Replicate Score The negative natural log of the enrichment p-value derived from the contingency table (Fisher s Exact test) or the Chi-square test For each factor in the data a separate score is calculated. This is derived from the negative log (base 10) of the average p-value for genes within the pathway for each factor, respectively. A high score indicates that the genes which fall into the pathway have a low p-value for the given factor (i.e. display a greater degree of differential expression) When assessing the results both the Enrichment Score and the Factor Score should be used. While you will intuitively want to sort by the Enrichment Score (indeed Pathway sorts by this column as the default option) this can sometimes be misleading. For example, if the number of genes in the entire pathway is small it only takes a few genes being differentially expressed to give a high Enrichment Score. If the same number of genes is differentially expressed in a pathway with many more genes the Enrichment Score will be lower. This is where the Factor Score can be particular informative: if two pathways have same similar Enrichment Scores, how can you interpret this? A higher Factor Score indicates a greater average degree of differential expression, thus this pathway is potentially more interesting.. Partek Pathway Quick Start Guide 6

7 Pathway The Project Elements pane displays the current pathway (in this case the most enriched pathway), the original gene list (significant genes between brain and heart), the Pathway Enrichment spreadsheet (as highlighted in Figure 6 above), and the Library References used (Figure 8). Figure 8: Project Elements In the event the Pathway libraries are not downloaded, click on the Pathway Libraries icon (circled in Figure 8) you can now download available libraries, or choose specific pathways from libraries that have already been downloaded (Figure 9). Figure 9: Manually adding Libraries/Pathways It is also easy to open additional pathway diagrams from the Enrichment table: Select the Pathway-Enrichment list (Figure 10) Partek Pathway Quick Start Guide 7

8 Figure 10: Opening additional pathways in the enrichment table Note: It is also possible to sort any of the tables within Pathway, simply by clicking on the column header. The first click will sort ascending, while the second will sort descending. Click on a Pathway ID to open the diagram (Figure 11) Figure 11: Viewing additional pathways The pathways can also be searched for known genes/genes of interest. Open the search tools be clicking the +/- button (bottom left corner of Figure 12) Select the species/libraries to be searched (note that in this example both mouse and human for both KEGG and Reactome are searchable) Type the search term (gene symbol) into the search box and press enter In the Search Results window all pathways containing the gene symbol will be shown Partek Pathway Quick Start Guide 8

9 Double-click on a pathway to open it Figure 12: Searching pathways for a gene of interest With a pathway diagram open it is now possible to colour the genes by various factors, such as p-value or fold-change see the Configuration pane in the upper right corner (Figure 13). By default the map is coloured by the first p-value ( Tissue in the this example) If you have checked to include the original ANOVA table during invoking Pathway (see above), you will the additional columns in the list below the Brain vs Heart columns. Figure 13: Colour by To illustrate, return to the Pathway Enrichment list and open the 6th most enriched pathway (Long-term potentiation) by double-clicking From the Colour By drop-down select Fold-Change the pathway will now be coloured (Figure 14) Partek Pathway Quick Start Guide 9

10 Figure 14: Pathway coloured by Fold-Change One gene in the pathway is up-regulated (green) this gene is AMPAr. To learn more about the gene, right click to invoke the contextual menu (Figure 15) you now have options to link out to KEGG, to External Links (Ensemble or UniProt), or to jump to the gene in the original list. (Note that the gene in the list is GRIA2 this is a synonym of AMPAr) Figure 15: Additional information Partek Pathway Quick Start Guide 10

11 Finally, the Pathway Detail pane (bottom left) show additional information relating to the selected pathway, and the genes within it (Figure 16) Figure 16: Pathway Elements End of Tutorial The steps covered in this tutorial are: Generating a Pathway Enrichment spreadsheet in GS Viewing the results in Partek Pathway Sorting and searching Pathway results For additional assistance, contact our technical support staff by phone at or by support@partek.com. Copyright 2011 by Partek Incorporated. All Rights Reserved. Reproduction of this material without express written consent from Partek Incorporated is strictly prohibited. Partek Pathway Quick Start Guide 11