BIOL398-01/S11:Week 12

UNDER CONSTRUCTION!

This journal entry is due on Tuesday, April 12 at midnight PDT (Wednesday night/Thursday morning). NOTE new due date and that the server records the time as Eastern Daylight Time (EDT). Therefore, midnight will register as 03:00.

Individual Journal Assignment

• Store this journal entry as "username Week 12" (i.e., this is the text to place between the square brackets when you link to this page).
• Create the following set of links. (HINT: you can do all of this easily by adding them to your template and then using the template on your pages.)
  • Link to your journal entry from your user page.
  • Link back from your journal entry to your user page.
  • Link to this assignment from your journal entry.
  • Don't forget to add the "BIOL398-01/S11" category to the end of your wiki page.

Background

This is a list of steps required to analyze DNA microarray data.

1. Quantitate the fluorescence signal in each spot
2. Calculate the ratio of red/green fluorescence
3. Log transform the ratios
4. Normalize the ratios on each microarray slide
   • Steps 1-4 are performed by the GenePix Pro software.
   • You will perform the following steps:
5. Normalize the ratios for a set of slides in an experiment
6. Perform statistical analysis on the ratios
7. Compare individual genes with known data
   • Steps 5-7 are performed in Microsoft Excel
8. Pattern finding algorithms (clustering)
9. Map onto biological pathways
   • We will use software called STEM for the clustering and mapping
   • We will use the YEASTRACT Database to determine which transcription factors are likely to regulate the genes in our clusters.
10. Create mathematical model of transcriptional network

We will perform steps 8-9 this week.

Clustering and Gene Ontology Analysis with STEM

1. Begin by downloading and extracting the STEM software. Click here to go to the STEM web site.
   • Click on the download link, register, and download the stem.zip file to your Desktop.
   • Unzip the file. In Seaver 120, you can right click on the file icon and select the menu item 7-zip > Extract Here.
   • This will create a folder called stem. Inside the folder, double-click on the stem.cmd to launch the STEM program.
2. Prepare your microarray data file for loading into STEM.
   • Using the Excel spreadsheet that you turned in for your Week 11 Assinment, insert a new worksheet and name it "stem".
   • Select all of the data from your "final" worksheet and paste it into your "stem" worksheet.
     • Your leftmost column should have the column header "MasterIndex". Rename this column to "SPOT". Column B should be named "ID". Rename this column to "Gene Symbol".
     • Delete all of the data columns EXCEPT for the AvgLogFC columns for each timepoint.
     • Rename the data columns with just the time and units. Use the same timepoints for all samples. For example, 15m, 30m, etc. for the Dahlquist lab data or 0.17h, 0.5h, etc. for the Schade data.
     • Save your work. Then use Save As to save this spreadsheet at Text (Tab-delimited) (*.txt). Click OK to the warnings and close your file.
3. Running STEM
   1. In section 1 of the the main STEM interface window, click on the Browse... button to navigate to and select your file.
      • Click on the radio button No normalization/add 0.
      • Check the box next to Spot IDs included in the data file.
   2. In section 2 of the main STEM interface window, select Saccharomyces cerevisiae (SGD), from the drop-down menu for Gene Annotation Source. Select No cross references, from the Cross Reference Source drop-down menu. Select No Gene Locations from the Gene Location Source drop-down menu.

Shared Journal Assignment

• Store your journal entry in the shared Class Journal Week 12 page. If this page does not exist yet, go ahead and create it (congratulations on getting in first :) )
• Link to your journal entry from your user page.
• Link back from the journal entry to your user page.
• Sign your portion of the journal with the standard wiki signature shortcut (~~~~).
• Add the "BIOL398-01/S11" category to the end of the wiki page (if someone has not already done so).

Reflection