Using two-way ANOVA to dissect the immune response to hookworm infection in mouse lung

The following tutorial walks through the identification of biological themes associated with genes that show a significant strain effect in a comparison of the response to hookworm infection in wild type and SCID mice. Genes with a significant strain effect are those that show a significant difference between the wild type and SCID mice regardless of whether there is any difference across the time series.

Before you begin, we recommend that you view the webinar. Select the The top button in the panel on the right to view the webinar.

When you are ready to begin this hands-on tutorial, click the third button down on the right, labeled "Log in to the dataset with tutorial."

1. Select "Projects" under the "Analysis" section in the "Control Panel" on the left side of the screen.

2. Select the magnifying glass icon next to the project labelled “Infection time series :: Filter :: Strain” in the list.

3. Select the “Cluster” link in the grey bar in the middle of the page.

4. Partition clustering will be used to separate the 517 genes into two clusters based on their expression patterns. Set clusters to "2" and distance to "correlation" and then click on the "Search" button in the "PAM-Partitioning around medoids" box.

5. The 517 genes have been split into 2 groups based on their expression patterns. Cluster 1 contains 334 genes that show lower expression in all of the SCID samples. Cluster 2 contains 183 genes that show higher expression in all of the SCID samples than in the wild type samples. Select the graph for Cluster 1. See the online help system for more information about the Silhouette Widths.

Note: To the view page-specific online help documents for any page, select the question mark icon located in the upper right corner of each page.

6. To view data and a gene summary for any gene in the list, click the Gene Name. This will bring up a data summary and a One-Click Gene Summary™ (OCGS) for the gene. The One-Click Gene Summary provides a synopsis of current UniGene and Entrez Gene information for the gene.

7. Select the Ontology link at the top of the screen to view a summary of the Gene Ontology terms associated with the genes in Cluster 1. See the online help system for information about the other reports.

8. The Ontology Report summarizes the Gene Ontology terms associated with the genes in Cluster 1. See the help documents for this page for more information about the Ontology Report.

9. Click on Z-score report in the upper right corner of the Ontology Report window.

10. The z-score report lists the biological process ontologies that are significantly over or under-represented in Cluster 1 (z-score greater than 2 or less than -2, respectively). Select the z-score link at the top of the last column to sort the ontologies by z-score.

11. The z-score report shows that there is a significant enrichment of genes involved in several biological processes, including B cell activation and T cell activation. Select the icon in the Genes column to view a list of the genes in Cluster 1 with this ontology.

Z-score reports can be generated for the Molecular Function and Cellular Component ontologies as well. Selecting the KEGG link will generate a similar report for KEGG pathway terms.


Only a few specific aspects of the data set have been explored here. You can also examine the ontologies associated with the genes in Cluster 2 and you can also cluster the significant time effect or interaction genes.