Bhamilton18 Week 10

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Electronic Notebook Steps

File Uploads

Excel Sheet: dHAP4 Excel Files

Stem Results Screenshots: STEM Profiles Powerpoint

Zip Files of genelist/GOlist and p-values: Genelist, GOlist and P-values Powerpoint

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: dHAP4 Excel Files
    • 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?
        • This profile contained the most variation in it's data as well as appears to have the largest difference among the genes invovled.
      • How many genes belong to this profile? 63.0
      • How many genes were expected to belong to this profile? 41.0
      • What is the p value for the enrichment of genes in this profile? 7.3 E-4
      • 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? 12
        • 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? 0
      • Select 6 Gene Ontology terms from your filtered list (either p < 0.05 or corrected p < 0.05). I used plain p-value
        • GO:2000112 regulation of cellular macromolecule biosynthetic process
        • GO:0071496 cellular response to external stimulus
        • GO:0009889 regulation of biosynthetic process
        • GO:0009605 response to external stimulus
        • GO:0032446 protein modification by small protein conjugation
        • GO:0019219 regulation of nucleobase-containing compound metabolic process
      • Look up the definitions for each of the terms at http://geneontology.org.
        • GO:2000112=> Any process that modulates the frequency, rate or extent of cellular macromolecule biosynthetic process. Source: GOC:obol
        • GO:0071496=> Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an external stimulus. Source: GOC:mah
        • GO:0009889=> Any process that modulates the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of substances. Source: GOC:go_curators
        • GO:0009605=> Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an external stimulus. Source: GOC:hb
        • GO:0032446=> A protein modification process in which one or more groups of a small protein, such as ubiquitin or a ubiquitin-like protein, are covalently attached to a target protein. Source: GOC:mah
        • GO:0019219=> Any cellular process that modulates the frequency, rate or extent of the chemical reactions and pathways involving nucleobases, nucleosides, nucleotides and nucleic acids. Source: GOC:go_curators
      • 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.

Conclusion Paragraph

During this assignment I was able to pinpoint significant gene strands within the dHAP4 data. I found 8 significant profiles, and followed their data in graph and GO table format. I focused primarily on the #2 profile for my dHAP4 data and found very few significant p-values (12) to analyze. I found profile #2 contains many reoccurring regulation GO terms within the remaining data, but focused and defined 6 of those terms. Profile #2 contained many variations within the data, whereas skimming the other profiles I found much more consistency and amount of data to be much higher. Overall, while I was unable to look at other data beyond my own, it appears dHAP4 had quite a large amount of significant profiles, but majority of those profiles appeared consistent.

Acknowledgments

  1. I worked with my partner Nicole Kalcic this week. We messaged each other with questions as well as met in person.
  2. My excel work was based off of data given to me by Dr. Dahlquist and Dr. Dionisio.
  3. All instructions came from the Week 10 instructions and were altered to fit my personal gene, dHAP4.
  4. All definitions were copied from the http://geneontology.org website.
  5. While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

References

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


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