Difference between revisions of "Aporras1 Week 10"

Revision as of 23:51, 29 November 2017

User page: Antonio Porras

Assignment page: Week 10 Assignment

Electronic Notebook

Clustering and GO Term Enrichment with stem

1. Prepare your microarray data file for loading into STEM.
   • Downloaded Excel workbook AP_dZAP1 from the Week 8 assignment.
   • Inserted a new worksheet into the Excel workbook, and named it "dZAP1_stem".
   • Selected all of the data from "dZAP1_ANOVA" worksheet and used Paste special > pasted values into "dZAP1_stem" worksheet.
     • Changed leftmost column header "Master_Index" to "SPOT". Changed column B "ID" to "Gene Symbol". Deleted the column named "Standard_Name".
     • Filtered the data on the B-H corrected p value to be > 0.05 (that's greater than in this case).
       • Selected all of the rows (except for the header row) and deleted the rows by right-clicking and choosing "Delete Row" from the context menu. Undid the filter. This ensured that I would cluster only the genes with a "significant" change in expression and not the noise. Record the number of genes left in your electronic notebook. 1775 genes were left.
     • Deleted all of the data columns EXCEPT for the Average Log Fold change columns for each timepoint (for example, wt_AvgLogFC_t15, etc.).
     • Renamed the data columns with just the time and units (for example, 15m, 30m, etc.).
     • Saved my work. Then used Save As to save the spreadsheet as Text (Tab-delimited) (*.txt). Clicked OK to the warnings and closed my file.
       • Turned on the file extensions.
2. Downloaded and extracted the STEM software. Click here to go to the STEM web site.
   • Clicked on the download link, registered, and downloaded the stem.zip file to the Desktop.
   • Unzipped the file.
   • This created a folder called stem. Inside the folder, double-clicked on the stem.jar and launched the STEM program.
3. Running STEM
   1. In section 1 (Expression Data Info) of the the main STEM interface window, clicked on the Browse... button to navigate to the file.
      • Clicked on the radio button No normalization/add 0.
      • Checked the box next to Spot IDs included in the data file.
   2. In section 2 (Gene Info) of the main STEM interface window, selected Saccharomyces cerevisiae (SGD), from the drop-down menu for Gene Annotation Source. Selected No cross references, from the Cross Reference Source drop-down menu. Selected No Gene Locations from the Gene Location Source drop-down menu.
   3. In section 3 (Options) of the main STEM interface window, made sure that the Clustering Method says "STEM Clustering Method" and didn't change the defaults for Maximum Number of Model Profiles or Maximum Unit Change in Model Profiles between Time Points.
   4. In section 4 (Execute) clicked on the yellow Execute button to run STEM.
4. Viewing and Saving STEM Results
   1. A new window opened 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.
      • Clicked 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:", clicked on the radio button that says "Based on real time". Then closed the Interface Options window.
      • Took 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 pasted it into a PowerPoint presentation to save my figures.
        • Uploaded Powerpoint to the wiki and linked them on my individual page as: AP dZAP STEM Screenshots.pptx
   2. Clicked 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.
      • Took a screenshot of each of the individual profile windows and saved the images in my 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, I clicked on the "Profile Gene Table" button to see the list of genes belonging to the profile. In the window that appears, clicked on the "Save Table" button and saved the file to my desktop.
        • Uploaded these files to the wiki and linked to them on my individual journal page.
          • dZAP1_profile7_genelist.txt
          • dZAP1_profile9_genelist.txt
          • dZAP1_profile22_genelist.txt
          • dZAP1_profile28_genelist.txt
          • dZAP1_profile40_genelist.txt
          • dZAP1_profile45_genelist.txt
          • dZAP1_profile48_genelist.txt
      • For each of the significant profiles, clicked 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 saved the file to my desktop.
        • Uploaded these files to the wiki and linked to them on my individual journal page.
          • dZAP1_profile7_GOlist.txt
          • dZAP1_profile9_GOlist.txt
          • dZAP1_profile22_GOlist.txt
          • dZAP1_profile28_GOlist.txt
          • dZAP1_profile40_GOlist.txt
          • dZAP1_profile45_GOlist.txt
          • dZAP1_profile48_GOlist.txt
5. Analyzing and Interpreting STEM Results
   1. Selected one of the profiles you saved in the previous step for further intepretation of the data. Answered the following:
      • Why did you select this profile? In other words, why was it interesting to you? I selected profile 22 because it had a decent amount of genes compared to the other profiles and a very low, significant, p-value.
      • How many genes belong to this profile? 252 genes belong to the profile.
      • How many genes were expected to belong to this profile? 26.6 genes expected to belong to the profile.
      • What is the p value for the enrichment of genes in this profile? The p-value is 2.0E-157 which is a very small p-value.
      • Opened the GO list file I saved for this profile in Excel. Selected the third row and then chose from the menu Data > Filter > Autofilter. Filtered on the "p-value" column to show only GO terms that have a p value of < 0.05. How many GO terms are associated with this profile at p < 0.05? 209 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? 27 GO terms are associated with this profile with a correct p-value < 0.05.
      • Selected 6 Gene Ontology terms from your filtered list (either p < 0.05 or corrected p < 0.05). Actin binding GO:0003779, Whole membrane GO:0098805, Mitochondrian GO:0005739, Detoxification GO:0098754, Cytoskeletal protein binding GO:0008092, Glucose 6-phosphate metabolic process GO:0051156.
        • Each member of the group will be reporting on his or her own cluster in your presentation next week. You should take care to choose terms that are the most significant, but that are also not too redundant.
          • Noted whether the same GO terms are showing up in multiple clusters.
        • Looked up the definitions for each of the terms at http://geneontology.org.
        • Went to http://geneontology.org to look up definitions.
        • Copied 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, clicked on the button that says "Link to detailed information about <term>, in this case "biological phase"".
        • The definition was found on the next results page, e.g. here.

Deliverable Files

1. Media:AP dZAP1 STEM Profiles and Powerpoint.zip

Acknowledgements

1. Worked in class with Katie Wright to discuss any questions we had throughout the process of completing the Week 10 assignment.
2. Recieved help from both Dondi and Dr. Dahlquist during the allotted class period.

While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

Aporras1 (talk) 14:53, 31 October 2017 (PDT)

References

1. LMU BioDB 2017. (2017). Week 10. Retrieved October 31, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_10