Aporras1 Week 10

From LMU BioDB 2017
Jump to: navigation, search

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. 1785 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 ID GO:0003779 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 actin binding".
        • The definition was found on the next results page.
          • Actin binding GO:0003779 - "Interacting selectively and non-covalently with monomeric or multimeric forms of actin, including actin filaments."
        • Copied and paste the ID GO:0098805 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 whole membrane".
        • The definition was found on the next results page.
          • Whole membrane GO:0098805 - "Any lipid bilayer that completely encloses some structure, and all the proteins embedded in it or attached to it. Examples include the plasma membrane and most organelle membranes."
        • Copied and paste the ID GO:0005739 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 mitochondrian".
        • The definition was found on the next results page.
          • Mitochondrian GO:0005739 - "A semiautonomous, self replicating organelle that occurs in varying numbers, shapes, and sizes in the cytoplasm of virtually all eukaryotic cells. It is notably the site of tissue respiration."
        • Copied and paste the ID GO:0098754 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 detoxification".
        • The definition was found on the next results page.
          • Detoxification GO:0098754 - "Any process that reduces or removes the toxicity of a toxic substance. These may include transport of the toxic substance away from sensitive areas and to compartments or complexes whose purpose is sequestration of the toxic substance."
        • Copied and paste the ID GO:0008092 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 cytoskeletal protein binding".
        • The definition was found on the next results page.
          • Cytoskeletal protein binding GO:0008092 - "Interacting selectively and non-covalently with any protein component of any cytoskeleton (actin, microtubule, or intermediate filament cytoskeleton)."
        • Copied and paste the ID GO:0051156 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 glucose 6-phosphate metabolic process".
        • The definition was found on the next results page.
          • Glucose 6-phosphate metabolic process GO:0051156 - "The chemical reactions and pathways involving glucose 6-phosphate, a monophosphorylated derivative of glucose with the phosphate group attached to C-6."
    2. Why does the cell react to cold shock by changing the expression of genes associated with these GO terms? The cell reacts to cold shock by changing expression of genes associated with these go terms in order to adapt to the environmental stress. The stress causes the cell to react and divert energy from processes that aren't necessary to survive in order to use that energy towards responding to the cold stress.

Summary Paragraph

In week 10's assignment we were only able to complete up until the analysis and interpretation of the stem results. However, we still gained a thorough understanding of the use of stem to analyze cold shock response in saccharomyces cerevisiae inasmuch that I selected a profile, profile 22, to specifically find genes and GO terms which were most significant in change of expression during cold shock. These specific GO terms defined prior in this assignment are areas of the cell in which energy may be direct towards or away from in order to respond to the stimulation. Specifically,the yeast may be redirecting energy away from processes which aren't necessary in stress response and towards expression of genes which aid in responding to the stress. As we've learned in recent class presentations, there are fewer genes expressed in response to cold shock compared to heat shock and there seems to be a trend towards downregulation of genes.

Deliverable Files

  1. Media:AP dZAP1 STEM Profiles and Powerpoint.zip
  2. Media:AP dZAP1 stem.xlsx
  3. Media:AP dZAP1 stem txt.txt

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.
  3. Instructions in electronic notebook were copied and modified from Week 10 assignment page.

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
  2. Short Time-series Expression Miner (STEM). (2006). Retrieved November 29, 2017, from http://www.cs.cmu.edu/~jernst/stem/
  3. Gene Ontology Consortium. (2017). The Gene Ontology. Retrieved November 29, 2017, from http://geneontology.org
  4. Glucose 6-phosphate metabolic process. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0000054
  5. Cytoskeletal protein binding. The Gene Ontology. Retrieved November 29, 2017, http://amigo.geneontology.org/amigo/term/GO:0008092
  6. Detoxification. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0098754
  7. Actin binding. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0003779
  8. Mitochondrion. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0005739
  9. Whole membrane. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0098805
Retrieved from "https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php?title=Aporras1_Week_10&oldid=5210"