Kmill104 Week 10 - Revision history

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 Except for what is noted above, this individual journal entry was completed by me and not copied from another source.
-[[User:Kmill104|Kmill104]] ([[User talk:Kmill104|talk]]) 22:41, 2 April 2024 (PDT)
+[[User:Kmill104|Kmill104]] ([[User talk:Kmill104|talk]]) 19:33, 10 April 2024 (PDT)
 ==References==

← Older revision		Revision as of 02:32, 11 April 2024
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	==Conclusion==		==Conclusion==
		+	During this week's journal assignment, I learned how to prepare and download microarray data into STEM. I also gained insight on how to run STEM, and how to view, save, and analyze the results I obtained from the software. I also learned how to use YEASTRACT and how it shows which transcription factors are significant for a given gene expression cluster. I used the list of significant factors I obtained from YEASTRACT to create my Gene Regulatory Network with GRNsight, and then learned how to generate and correct the input workbook for this software. Finally, I worked throughout this process to keep an organized and detailed lab notebook, so that any person could follow these steps and get the same results.

	==Acknowledgements==		==Acknowledgements==

← Older revision		Revision as of 02:22, 11 April 2024
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	==Purpose==		==Purpose==
		+	The purpose of this week's journal assignment was to analyze a DNA microarray dataset through "high level" analysis. This included using the STEM software for clustering, YEASTRACT for finding which transcription factors regulate these gene clusters, and producing a gene regulatory network in GRNsight. We also worked to keep an organized and detail laboratory notebook, so that another person could follow these steps and reproduce the same results.

	==Methods/Results==		==Methods/Results==

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 ==Data & Files==
-Your data and files section should include:
-* Your Excel workbook with all of your calculations. [[Media:BIOL367_S24_microarray-data_wt_KM2.xlsx | KM WT Workbook]]
+* [[Media:BIOL367_S24_microarray-data_wt_KM2.xlsx | KM WT Workbook]]
-** Your YEASTRACT results should be saved as a new worksheet in this workbook.
+* [[Media:KM_wt_slide.pptx | KM PowerPoint Slide]]
-** Note that you will be working with this workbook for the next week or two, adding computations to it.  Save the <u>new versions</u> to the wiki with the '''same filename'''.  The wiki will store each version of the file so you can always go back to a previous version, if need be.
+* [[Media:KM_wt_STEM.txt | KM STEM .txt File]]
-* Your PowerPoint slide with a summary table of p values, <u>updated with the screenshots from the stem software</u>. [[Media:KM_wt_slide.pptx | KM PowerPoint Slide]]
+* [[Media:WT_Profile_GeneList.zip]] [[Media:WT_Profile_GOList.zip]]
-** You will also be adding to the PowerPoint presentation during subsequent steps in the analysis.
+* [[Media:KM_GRN2.png | Gene Regulatory Network KM]]
-* The input .txt file that you used to run stem. [[Media:KM_wt_STEM.txt | KM STEM .txt File]]
+* [[Media:KM_GRN2.xlsx | Excel Workbook for GRN KM]]
 ==Conclusion==

@@ Line 11: / Line 11: @@
 #** I then renamed the data columns with just the time and units, so for any column with wt_AvgLogFC_t#, the header became #m.
 #** I saved my work.  Then, I used ''Save As'' to save this spreadsheet as Text (Tab-delimited) (*.txt), clicked OK to the warnings and closed my file.
-# '''Then, I downloaded and extracted the STEM software.''' [http://www.cs.cmu.edu/~jernst/stem/ Click here to go to the STEM web site].
+# '''Then, I downloaded and extracted the STEM software.'''
 #* I clicked on the [http://www.sb.cs.cmu.edu/stem/stem.zip download link] and downloaded the <code>stem.zip</code> file to my Desktop.
 #* I extracted the file by right-clicking on it and selecting "Extract all" from the menu.
 #* This created a folder called <code>stem</code>.
 #** I then downloaded the Gene Ontology and yeast GO annotations, which I placed in this same folder. I used [https://lmu.box.com/s/t8i5s1z1munrcfxzzs7nv7q2edsktxgl "gene_ontology.obo"] to download the file for Gene Ontology.
 #** I used [https://lmu.box.com/s/zlr1s8fjogfssa1wl59d5shyybtm1d49 "gene_association.sgd.gz"] to download the yeast GO annotations.

@@ Line 11: / Line 11: @@
 #** I then renamed the data columns with just the time and units, so for any column with wt_AvgLogFC_t#, the header became #m.
 #** I saved my work.  Then, I used ''Save As'' to save this spreadsheet as Text (Tab-delimited) (*.txt), clicked OK to the warnings and closed my file.
-# '''Now, I downloaded and extract the STEM software.''' [http://www.cs.cmu.edu/~jernst/stem/ Click here to go to the STEM web site].
+# '''Then, I downloaded and extracted the STEM software.''' [http://www.cs.cmu.edu/~jernst/stem/ Click here to go to the STEM web site].
 #* I clicked on the [http://www.sb.cs.cmu.edu/stem/stem.zip download link] and downloaded the <code>stem.zip</code> file to my Desktop.
 #* I extracted the file by right-clicking on it and selecting "Extract all" from the menu.
@@ Line 20: / Line 20: @@
 #*Inside the folder, I double-clicked on the <code>stem.jar</code> to launch the STEM program.
 # '''Running STEM'''
-## 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.
+## In section 1 (Expression Data Info) of the the main STEM interface window, I clicked on the ''Browse...'' button to navigate to and select my file.
-##* Click on the radio button ''No normalization/add 0''.
+##* I then clicked on the radio button ''No normalization/add 0''.
 ##* Check the box next to ''Spot IDs included in the data file''.
-## In section 2 (Gene Info) of the main STEM interface window, leave the default selection for the three drop-down menu selections for Gene Annotation Source, Cross Reference Source, and Gene Location Source as "User provided".
+## In section 2 (Gene Info) of the main STEM interface window, I left the default selection for the three drop-down menu selections for Gene Annotation Source, Cross Reference Source, and Gene Location Source as "User provided".
-## Click the "Browse..." button to the right of the "Gene Annotation File" item.  Browse to your "stem" folder and select the file "gene_association.sgd.gz" and click Open.
+## I clicked the "Browse..." button to the right of the "Gene Annotation File" item.  I then browsed to my "stem" folder and selected the file "gene_association.sgd.gz" and clicked Open.
-## 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.
+## In section 3 (Options) of the main STEM interface window, I checked that the Clustering Method says "STEM Clustering Method" and did not change the defaults for Maximum Number of Model Profiles or Maximum Unit Change in Model Profiles between Time Points.
-## In section 4 (Execute) click on the yellow Execute button to run STEM.
+## In section 4 (Execute), I clicked on the yellow Execute button to run STEM.
 # '''Viewing and Saving STEM Results'''
-## 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.
+## A new window opened called "All STEM Profiles (1)".  Each box corresponded to a model expression profile.  Colored profiles had a statistically significant number of genes assigned; they were arranged in order from most to least significant p value.  Profiles with the same color belonged to the same cluster of profiles.  The number in each box was 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.
+##* I 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:", I clicked on the radio button that says "Based on real time".  I then closed the Interface Options window.
-##*Take a screenshot of this window (on a PC, simultaneously press the <code>Alt</code> and <code>PrintScreen</code> buttons to save the view in the active window to the clipboard) and paste it into a PowerPoint presentation to save your figures.
+##*I took a screenshot of this window, and then pasted it into a PowerPoint presentation that also contained my p-value table.
-## 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.
+## I then clicked on each of the SIGNIFICANT profiles 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.
+##* I 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, 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.
+##* 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 appeared, I clicked on the "Save Table" button and saved the file to my desktop. I named the file "wt_profile#_genelist.txt", where I replaced 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.  To do this, select all of the files you want to zip together.  Then right click and select "Send to" and "Compressed (zipped) folder" from the context menu.
+##** I then zipped these files together, and then uploaded these zipped files to the wiki and linked them to this page.
-##* 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!
+##* For each of the significant profiles, I clicked on the "Profile GO Table" to see the list of Gene Ontology terms belonging to the profile.  In the window that appeared, I clicked on the "Save Table" button and saved the file to your desktop.  I made the filename "wt_profile#_GOlist.txt" and replaced 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.  To do this, select all of the files you want to zip together.  Then right click and select "Send to" and "Compressed (zipped) folder" from the context menu.
+##** Again, I zipped these files together, and then uploaded these zipped files to the wiki and linked them to this page.
 # '''Analyzing and Interpreting STEM Results'''

@@ Line 2: / Line 2: @@
 ==Methods/Results==
-# '''Prepare your microarray data file for loading into STEM.'''
+# '''For this this week's journal assignment, my first step was to prepare my microarray data file for loading into STEM.'''
-#* Insert a new worksheet into your Excel workbook, and name it "(STRAIN)_stem".
+#* I inserted a new worksheet into my Excel workbook, and named it "wt_stem".
-#* Select all of the data from your "(STRAIN)_ANOVA" worksheet and Paste special > paste values into your "(STRAIN)_stem" worksheet.
+#* I then selected all of the data from my "wt_ANOVA" worksheet and Paste special > pasted values into my "wt_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".
+#** I then renamed the column with the header "Master_Index" to "SPOT". I also renamed the column with the column header "ID" to "Gene Symbol". I deleted the column with the header "Standard_Name".
-#** Filter the data on the B-H corrected p value to be > 0.05 (that's '''greater than''' in this case).
+#** I filtered the data on the B-H corrected p value column to be > 0.05.
-#*** 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.
+#***Once the data had been filtered, I selected all of the rows (except for my header row) and deleted the rows by right-clicking and choosing "Delete Row" from the context menu.  I then undid the filter.  This step ensured that the genes I will cluster will only the genes that had a "significant" change in expression.
-#** Delete all of the data columns '''''EXCEPT''''' for the Average Log Fold change columns for each timepoint (for example, wt_AvgLogFC_t15, etc.).
+#** I then deleted all of the other data columns '''''EXCEPT''''' for the Average Log Fold change columns for each timepoint.
-#** Rename the data columns with just the time and units (for example, 15m, 30m, etc.).
+#** I then renamed the data columns with just the time and units, so for any column with wt_AvgLogFC_t#, the header became #m.
-#** If you had #DIV/0! errors remaining, it will cause problems. To remove these, open the Find/Replace dialog.  Search for #DIV/0!, but don't put anything in the replace field.  Click "Replace all" to remove the #DIV/0! errors.
+#** I saved my work.  Then, I used ''Save As'' to save this spreadsheet as Text (Tab-delimited) (*.txt), clicked OK to the warnings and closed my file.
-#** 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.
+# '''Now, I downloaded and extract the STEM software.''' [http://www.cs.cmu.edu/~jernst/stem/ Click here to go to the STEM web site].
-#*** Note that you should turn on the file extensions if you have not already done so.
+#* I clicked on the [http://www.sb.cs.cmu.edu/stem/stem.zip download link] and downloaded the <code>stem.zip</code> file to my Desktop.
-# '''Now download and extract the STEM software.'''  [http://www.cs.cmu.edu/~jernst/stem/ Click here to go to the STEM web site].
+#* I extracted the file by right-clicking on it and selecting "Extract all" from the menu.
-#* Click on the [http://www.sb.cs.cmu.edu/stem/stem.zip download link] and download the <code>stem.zip</code> file to your Desktop.
+#* This created a folder called <code>stem</code>.
 #** You now need to download the Gene Ontology and yeast GO annotations and place them in this folder.
-#** Click here to download the file [https://lmu.box.com/s/t8i5s1z1munrcfxzzs7nv7q2edsktxgl "gene_ontology.obo"].
+#** I then downloaded the Gene Ontology and yeast GO annotations, which I placed in this same folder. I used [https://lmu.box.com/s/t8i5s1z1munrcfxzzs7nv7q2edsktxgl "gene_ontology.obo"] to download the file for Gene Ontology.
-#** Click here to download the file [https://lmu.box.com/s/zlr1s8fjogfssa1wl59d5shyybtm1d49 "gene_association.sgd.gz"].
+#** I used [https://lmu.box.com/s/zlr1s8fjogfssa1wl59d5shyybtm1d49 "gene_association.sgd.gz"] to download the yeast GO annotations.
-#*Inside the folder, double-click on the <code>stem.jar</code> to launch the STEM program.
+#*Inside the folder, I double-clicked on the <code>stem.jar</code> to launch the STEM program.
 # '''Running STEM'''
 ## 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.

@@ Line 46: / Line 46: @@
 ##* '''''How many genes belong to this profile?''''' 549.0
 ##* '''''How many genes were expected to belong to this profile?''''' 47.1
-##* '''''What is the p value for the enrichment of genes in this profile?''''' 1.00
+##* '''''What is the p value for the enrichment of genes in this profile?''''' 0.00
 ##**Bear in mind that we just finished computing p values to determine whether each individual gene had a significant change in gene expression at each time point.  This p value determines whether the number of genes that show this particular expression profile across the time points is significantly more than expected.
 ## 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 third row and then choose from the menu Data > Filter > Autofilter.  Filter 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?''''' 81