This tutorial will take you through the analysis of one of the demo lists hosted on DAVID. The data needed for this analysis are included in all of the "human" distributions of EASE.

1. Start EASE by double-clicking the EASE icon in the \EASE\ directory.

2. Take notice of the layout of the opening screen. At the top are buttons for help files, etc. (Most importantly, take note of the e-mail address 'ease@niaid.nih.gov' in case you find a bug in the program or have questions not answered on the DAVID website.) Farther down is the [update with the most recent online data] button. This launches a process to download flat files from various sources and install the files that EASE needs. Farther down is the SELECT OUTPUT area. Here you can specify analysis to be saved as files, or just displayed in the web browser. The next two sections are most important: The INPUT GENES section is where you enter the gene list you wish to study. The EXPLORE section launches the three basic processes of EASE to explore your list.

3. Make sure "Affymetrix probesets" is displayed in the drop-down menu in the INPUT GENES section.

4. Click [from a text file] in the INPUT GENES section.

5. Select the file "Cicala Up with HIV Env exposure.txt" and click [open]. These are genes from an Affymetrix GeneChip experiment that were up-regulated in PBMCs upon exposure to HIV envelope proteins.

6. Note that the gene identifiers have been loaded into the window.

7. Now use the tools in the EXPLORE section. Make sure "DAVID" is displayed in the drop-down menu in the EXPLORE section and click the [Link to:] button. Your systems default browser should launch and take you to the DAVID website with your genes already loaded. Explore your gene list on DAVID if you have time.

8. Click the [Find over-represented gene categories] button. This will take you to a new window for specifying the population of genes assayed. This is equivalent to asking what all the genes on your microarray are.

9. Again make sure that "Affymetrix probesets" is displayed in the drop down box, and click [from a population file].

10. This experiment was run on the Affymetrix HG-U95Av2 GeneChip. So select the "HG-U95Av2.txt" file and click [open].

11. Notice the identifiers loaded into the window. Now you're ready for the analysis.

12. Click [run basic analysis]. This launches the process of loading all gene categorical information and calculating over-representation statistics. When these calculations are complete, the program will create an html table of the results in the \Ease\Temp\ directory and attempt to launch the systems default web browser to display it.

13. Look at the results. The fields are defined in the "over-representation" help file. For now, suffice it to say that the Gene Category at the top of the list is the most over-represented category in the gene list we analyzed. Now select the "Affymetrix probesets" for that category and right-click-copy them.

14. Close the browser window (if you want) and return to the EASE window.

15. Now we want to annotate the genes you just copied from that category. In the INPUT GENES section, click [clear all] to clear the old list and click [paste] to load the new sublist.

16. Click the [annotate genes] button. Now EASE will load annotation data, write this info to an HTML file in the \Ease\Temp\ directory and attempt to launch the system default web browser to view it.

...That's the tip of the iceberg. Explore the various functions and options in EASE. Also explore the directory structure and the files therein. EASE allows you to create and use your own fields for annotation, your own ways of categorizing genes, your own links to online tools, your own system of gene identifiers, your own population files to describe your microarray, etc. All of these files are simple text files, so they are easy to create and customize. Refer to the help file called "Customizing EASE.txt" in the "Advanced topics" folder for more information on all the things that can be customized with EASE. Happy story-hunting!