Nicolekalcic Week 10

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File:DHAP4nk.xlsx

File:DHAP4stemNK.txt

Electronic Journal

Deliverables Excel Sheet: Stem Results Screenshots: Zip Files of genelist/GOlist and p-values:

Clustering and GO Term Enrichment with stem

  1. Prepare your microarray data file for loading into STEM.
    • Download Excel workbook from our Week 8 assignment: Media:file
    • Insert a new worksheet into your Excel workbook, and name it "dHAP4_stem".
    • Select all of the data from "dHAP4_ANOVA" worksheet and Paste special > paste values into your "dHAP4_stem" worksheet.
      • Your leftmost column should have the column header "Master_Index". Rename this column to "SPOT". Column B should be named "ID". Rename this column to "Gene Symbol". Delete the column named "Standard_Name".
      • Filter the data on the B-H corrected p value to be > 0.05 (that's greater than in this case).
        • Once the data has been filtered, select all of the rows (except for your header row) and delete the rows by right-clicking and choosing "Delete Row" from the context menu. Undo the filter. This ensures that we will cluster only the genes with a "significant" change in expression and not the noise. There should be 1892 genes remaining.
      • Delete all of the data columns EXCEPT for the Average Log Fold change columns for each timepoint (for example, wt_AvgLogFC_t15, etc.).
      • Rename the data columns with just the time and units (for example, 15m, 30m, etc.).
      • Save your work. Then use Save As to save this spreadsheet as Text (Tab-delimited) (*.txt). Click OK to the warnings and close your file.
        • Note that you should turn on the file extensions if you have not already done so.
  2. Now download and extract 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.jar to launch the STEM program.
  3. Running STEM
    1. In section 1 (Expression Data Info) 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 (Gene Info) 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.
    3. In section 3 (Options) of the main STEM interface window, make sure that the Clustering Method says "STEM Clustering Method" and do not change the defaults for Maximum Number of Model Profiles or Maximum Unit Change in Model Profiles between Time Points.
    4. In section 4 (Execute) click on the yellow Execute button to run STEM.
  4. Viewing and Saving STEM Results
    1. A new window will open called "All STEM Profiles (1)". Each box corresponds to a model expression profile. Colored profiles have a statistically significant number of genes assigned; they are arranged in order from most to least significant p value. Profiles with the same color belong to the same cluster of profiles. The number in each box is simply an ID number for the profile.
      • Click on the button that says "Interface Options...". At the bottom of the Interface Options window that appears below where it says "X-axis scale should be:", click on the radio button that says "Based on real time". Then close the Interface Options window.
      • Take a screenshot of this window (on a PC, simultaneously press the Alt and PrintScreen buttons to save the view in the active window to the clipboard) and paste it into a PowerPoint presentation to save your figures.
    2. Click on each of the SIGNIFICANT profiles (the colored ones) to open a window showing a more detailed plot containing all of the genes in that profile.
      • Take a screenshot of each of the individual profile windows and save the images in your PowerPoint presentation.
      • At the bottom of each profile window, there are two yellow buttons "Profile Gene Table" and "Profile GO Table". For each of the profiles, click on the "Profile Gene Table" button to see the list of genes belonging to the profile. In the window that appears, click on the "Save Table" button and save the file to your desktop. Make your filename descriptive of the contents, e.g. "wt_profile#_genelist.txt", where you replace the number symbol with the actual profile number.
        • Upload these files to the wiki and link to them on your individual journal page. (Note that it will be easier to zip all the files together and upload them as one file).
      • For each of the significant profiles, click on the "Profile GO Table" to see the list of Gene Ontology terms belonging to the profile. In the window that appears, click on the "Save Table" button and save the file to your desktop. Make your filename descriptive of the contents, e.g. "wt_profile#_GOlist.txt", where you use "wt", "dGLN3", etc. to indicate the dataset and where you replace the number symbol with the actual profile number. At this point you have saved all of the primary data from the STEM software and it's time to interpret the results!
        • Upload these files to the wiki and link to them on your individual journal page. (Note that it will be easier to zip all the files together and upload them as one file).
  5. Analyzing and Interpreting STEM Results
    1. Select one of the profiles you saved in the previous step for further interpretation of the data. Profile #2
      • Why did you select this profile? In other words, why was it interesting to you?
      • How many genes belong to this profile?
      • How many genes were expected to belong to this profile?
      • What is the p value for the enrichment of genes in this profile?
      • Open the GO list file you saved for this profile in Excel. This list shows all of the Gene Ontology terms that are associated with genes that fit this profile. Select the seventh column and then choose from the menu Data > Filter > Number Filters > Less Than 0.05. How many GO terms are associated with this profile at p < 0.05?
        • The GO list also has a column called "Corrected p-value". This correction is needed because the software has performed thousands of significance tests. Filter on the "Corrected p-value" column to show only GO terms that have a corrected p value of < 0.05. How many GO terms are associated with this profile with a corrected p value < 0.05?
      • Select 6 Gene Ontology terms from your filtered list (either p < 0.05 or corrected p < 0.05).
      • Look up the definitions for each of the terms at http://geneontology.org.
      • Copy and paste the GO ID (e.g. GO:0044848) into the search field at center top of the page called "Search GO Data".
      • In the results page, click on the button that says "Link to detailed information about <term>, in this case "biological phase"".
      • The definition will be on the next results page, e.g. here.

Summary Paragraph

Acknowledgements

  1. I worked with my homework partner Blair Hamilton in class and via text.
  2. I used the template of the procedure on the Week 10 page to complete my electronic journal, modifying it to fit the strand dHAP4.
  3. All definitions were copied from the http://geneontology.org website.
  4. Dr. Dahlquist and Dr. Dionisio provided the data that I used for this assignment.

While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source. Nicolekalcic (talk) 14:57, 2 November 2017 (PDT)

References

Links

Nicole Kalcic's Homepage


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