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Association Analysis for Large-Scale Gene Set Data 25Some fields would contain more than one entry. For example, a genemight have alternative transcripts, in which case there would be more thanone RefSeq accession number, which would be separated by three rightslashes (///). Another exception is the chromosome coordinates columnwherein the fields (chromosome number, start, end, and orientation) are separatedwith three colons (:::).3.3. GO Association Analysis (GOTree Machine and the WebGestaltGO Module)All software packages listed in Table 1 could analyze associations betweengene sets and GO categories. There are several important considerations whenchoosing the tool that might be most appropriate for the analysis: scope of theanalysis (available organisms, GO levels), statistical approach (including referencegene set options), visualization options (interactive vs noninteractive), andupdate schedule (as almost all tools use database integration). There are otheradditional features that are not critical for the analysis, but might be of somebenefit to the user (see the detailed comparison at http://bioinfo.vanderbilt.edu/mpaper/tc.html).One could use the following steps to accomplish the analysis through theGOTree module in WebGestalt (see Note 5 on the differences betweenGOTree Machine (GOTM) and WebGestalt GO module and Note 6 on somerestrictions):1. Retrieve the “brain embryo imprint” gene set.2. Press the GOTREE button.3. Choose appropriate reference set and statistical approach (to observe some importantconsiderations on this choice, see Note 7). In this case, “WEBGESTALT_MG_U74AV2” and “Hypergeometric test.” are selected. It may take a few minutesbefore the calculations are done.4. Once the analysis process is completed, a new button CHECK GOTREE willappear. After pressing the button, an expandable GOTree will appear, with significantlyenriched GO categories highlighted.5. Press the direct acyclic graph (DAG) button (Fig. 1) for an enriched GO DAG, theBAR CHART button for a bar chart at the specified annotation level, or theEXPORT GOTREE button for a text output of the complete tree.6. To change the GO level and the main tree for a bar chart branch use the pull-downmenu under the “Bar chart” button (see explanation in Note 8).7. To export the intersection of the input gene set and a specific category follow oneof these steps:a. From the bar chart: press BAR CHART (opens new window), choose an interestingcategory and press the bar. This opens a new window with genes fromthe gene set being analyzed that are also in the selected category.