Difference between revisions of "Aporras1 Week 10"

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==Electronic Notebook==
 
==Electronic Notebook==
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This is a list of steps required to analyze DNA microarray data.
 
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#Quantitate the fluorescence signal in each spot
 
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#Calculate the ratio of red/green fluorescence
 
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#Log<sub>2</sub> transform the ratios
 
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#* Steps 1-3 have been performed for you by the GenePix Pro software (which runs the microarray scanner).
 
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#Normalize the ratios on each microarray slide
 
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#Normalize the ratios for a set of slides in an experiment
 
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#* Steps 4-5 was performed for you using a script in R, a statistics package (see: [https://openwetware.org/wiki/Dahlquist:Microarray_Data_Analysis_Workflow#Steps_4-5:_Within-_and_Between-chip_Normalization Microarray Data Analysis Workflow])
 
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#* You will perform the following steps:
 
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#Perform statistical analysis on the ratios
 
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#Compare individual genes with known data
 
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#* Steps 6-7 are performed in Microsoft Excel
 
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#Pattern finding algorithms (clustering)
 
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#Map onto biological pathways
 
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#* We will use software called STEM for the clustering and mapping
 
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# Identifying regulatory transcription factors responsible for observed changes in gene expression
 
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# Dynamical systems modeling of the gene regulatory network ([http://kdahlquist.github.io/GRNmap/ GRNmap])
 
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# Viewing modeling results in [http://dondi.github.io/GRNsight/ GRNsight]
 
  
 
==== Clustering and GO Term Enrichment with stem ====
 
==== Clustering and GO Term Enrichment with stem ====
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#* Selected all of the data from "dZAP1_ANOVA" worksheet and used Paste special > pasted values into "dZAP1_stem" worksheet.
 
#* 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".
 
#** Changed leftmost column header "Master_Index" to "SPOT".  Changed column B "ID" to "Gene Symbol".  Deleted the column named "Standard_Name".
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#** Filtered the data on the B-H corrected p value to be > 0.05 (that's '''greater than''' in this case).
 
#** Filtered the data on the B-H corrected p value to be > 0.05 (that's '''greater than''' in this case).
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#*** 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.
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#*** 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.).
 
#** 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.).
 
#** 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.
 
#** 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.
 
#*** Turned on the file extensions.
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# '''Now download and extract the STEM software.'''  [http://www.cs.cmu.edu/~jernst/stem/ Click here to go to the STEM web site].
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# '''Downloaded and extracted the STEM software.'''  [http://www.cs.cmu.edu/~jernst/stem/ Click here to go to the STEM web site].
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#* Click on the [http://www.andrew.cmu.edu/user/zivbj/stemreg.html download link], register, and download the <code>stem.zip</code> file to your Desktop.
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#* Clicked on the [http://www.andrew.cmu.edu/user/zivbj/stemreg.html download link], registered, and downloaded the <code>stem.zip</code> file to the Desktop.
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#* Unzip the file.  In Seaver 120, you can right click on the file icon and select the menu item ''7-zip > Extract Here''.
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#* Unzipped the file.  
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#* This will create a folder called <code>stem</code>.  Inside the folder, double-click on the <code>stem.jar</code> to launch the STEM program.
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#* This created a folder called <code>stem</code>.  Inside the folder, double-clicked on the <code>stem.jar</code> and launched the STEM program.
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<!--#** In Seaver 120, we encountered an issue where the program would not launch on the Windows XP machines due to a lack of memory. (Even though the computers have been upgraded to Windows 7, do this to launch the program.)  To get around this problem, launch STEM from the command line.
 
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#*** Go to the start menu and click on ''Programs > Accessories > Command Prompt''.
 
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#*** You will need to navigate to the directory (folder) in which the STEM program resides. If you followed the instructions above and extracted the stem folder to the Desktop, type the following:  <code>cd Desktop\stem</code>  and press "Enter".
 
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#*** To launch the program then type:  <code>java -mx512M -jar stem.jar -d defaults.txt</code>  and press "Enter".  This will launch the program with less memory allocated to it.-->
 
 
# '''Running STEM'''
 
# '''Running STEM'''
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## 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.
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## In section 1 (Expression Data Info) of the the main STEM interface window, clicked on the ''Browse...'' button to navigate to the file.
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##* Click on the radio button ''No normalization/add 0''.
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##* Clicked on the radio button ''No normalization/add 0''.
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##* Check the box next to ''Spot IDs included in the data file''.
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##* Checked the box next to ''Spot IDs included in the data file''.
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## 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.
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## 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.
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## 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.
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## 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.
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## In section 4 (Execute) click on the yellow Execute button to run STEM.
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## In section 4 (Execute) clicked on the yellow Execute button to run STEM.
 
# '''Viewing and Saving STEM Results'''
 
# '''Viewing and Saving STEM Results'''
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## 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.
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## 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.
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##* 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.
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##* 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.
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##*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.
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##*Took 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 pasted it into a PowerPoint presentation to save my figures.
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## 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.
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##**Uploaded Powerpoint to the wiki and linked them on my individual page as: AP dZAP STEM Screenshots.pptx
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##* Take a screenshot of each of the individual profile windows and save the images in your PowerPoint presentation.
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## 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.
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##* 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.
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##* Took a screenshot of each of the individual profile windows and saved the images in my PowerPoint presentation.
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##** 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).
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##* 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.
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##* 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!
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##** Uploaded these files to the wiki and linked to them on my individual journal page.  
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##** 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).
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##***dZAP1_profile7_genelist.txt
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##***dZAP1_profile9_genelist.txt
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##***dZAP1_profile22_genelist.txt
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##***dZAP1_profile28_genelist.txt
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##***dZAP1_profile40_genelist.txt
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##***dZAP1_profile45_genelist.txt
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##***dZAP1_profile48_genelist.txt
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##* 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
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##***dZAP1_profile9_GOlist.txt
 +
##***dZAP1_profile22_GOlist.txt
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##***dZAP1_profile28_GOlist.txt
 +
##***dZAP1_profile40_GOlist.txt
 +
##***dZAP1_profile45_GOlist.txt
 +
##***dZAP1_profile48_GOlist.txt
 
# '''Analyzing and Interpreting STEM Results'''
 
# '''Analyzing and Interpreting STEM Results'''
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## Select '''''one''''' of the profiles you saved in the previous step for further intepretation of the data.  I suggest that you choose one that has a pattern of up- or down-regulated genes at the cold shock timepoints.  '''''Each member of your group should choose a different profile.'''''  Answer the following:
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## Selected '''''one''''' of the profiles you saved in the previous step for further intepretation of the data.  Answered the following:
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##* '''''Why did you select this profile?  In other words, why was it interesting to you?'''''
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##* '''''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.
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##* '''''How many genes belong to this profile?'''''
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##* '''''How many genes belong to this profile?''''' 252 genes belong to the profile.
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##* '''''How many genes were expected to belong to this profile?'''''
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##* '''''How many genes were expected to belong to this profile?''''' 26.6 genes expected to belong to the profile.
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##* '''''What is the p value for the enrichment of genes in this profile?''''' 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.
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##* '''''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.
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##* 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?''''' 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?'''''
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##* 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.
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##* Select 6 Gene Ontology terms from your filtered list (either p < 0.05 or corrected p < 0.05).
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##* 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.
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##** 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.  For example, "RNA metabolism" and "RNA biosynthesis" are redundant with each other because they mean almost the same thing.
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##** 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.   
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##*** Note whether the same GO terms are showing up in multiple clusters.
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##*** Noted whether the same GO terms are showing up in multiple clusters.
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##**'''''Look up the definitions for each of the terms at [http://geneontology.org http://geneontology.org]. In your final presentation, you will discuss the biological interpretation of these GO terms. In other words, why does the cell react to cold shock by changing the expression of genes associated with these GO terms?  Also, what does this have to do with the transcription factor being deleted (for the Δgln3 and Δswi4 groups)?'''''
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##**'''''Looked up the definitions for each of the terms at [http://geneontology.org http://geneontology.org].  
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##** To easily look up the definitions, go to [http://geneontology.org http://geneontology.org].
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##** Went to [http://geneontology.org http://geneontology.org] to look up definitions.
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##** Copy and paste the GO ID (e.g. GO:0044848) into the search field at center top of the page called "Search GO Data".
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##** Copied and paste the ID GO:0003779 into the search field at center top of the page called "Search GO Data".
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##** In the [http://amigo.geneontology.org/amigo/medial_search?q=GO%3A0044848 results] page, click on the button that says "Link to detailed information about <term>, in this case "biological phase"".  
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##** In the results page, clicked on the button that says "Link to detailed information about actin binding".
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##** The definition will be on the next results page, e.g. [http://amigo.geneontology.org/amigo/term/GO:0044848 here].
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##** The definition was found on the next results page.
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##*** '''Actin binding GO:0003779''' - "Interacting selectively and non-covalently with monomeric or multimeric forms of actin, including actin filaments."
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==== Using YEASTRACT to Infer which Transcription Factors Regulate a Cluster of Genes ====
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##** 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.
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##*** '''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".
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##** 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.
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##*** '''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."
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##** Copied and paste the ID GO:0098754 into the search field at center top of the page called "Search GO Data".
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##** 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.
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##*** '''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."
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##** 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."
 +
## '''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.
  
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In the previous analysis using STEM, we found a number of gene expression profiles (aka clusters) which grouped genes based on similarity of gene expression changes over time. The implication is that these genes share the same expression pattern because they are regulated by the same (or the same set) of transcription factors. We will explore this using the YEASTRACT database.
+
==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.
  
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# Open the gene list in Excel for the one of the significant profiles from your stem analysis.  Choose a cluster with a clear cold shock/recovery up/down or down/up pattern.  You should also choose one of the largest clusters.
+
==Deliverable Files==
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#* Copy the list of gene IDs onto your clipboard.
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#[[Media:AP dZAP1 STEM Profiles and Powerpoint.zip]]
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# Launch a web browser and go to the [http://www.yeastract.com/ YEASTRACT database].
+
#[[Media:AP dZAP1 stem.xlsx]]
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#* On the left panel of the window, click on the link to [http://www.yeastract.com/formrankbytf.php ''Rank by TF''].
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#[[Media:AP dZAP1 stem txt.txt]]
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#* Paste your list of genes from your cluster into the box labeled ''ORFs/Genes''.
 
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#* Check the box for ''Check for all TFs''.
 
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#* Accept the defaults for the Regulations Filter (Documented, DNA binding plus expression evidence)
 
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#* Do '''''not''''' apply a filter for "Filter Documented Regulations by environmental condition".
 
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#* Rank genes by TF using: The % of genes in the list and in YEASTRACT regulated by each TF.
 
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#* Click the ''Search'' button.
 
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# Answer the following questions:
 
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#* In the results window that appears, the p values colored green are considered "significant", the ones colored yellow are considered "borderline significant" and the ones colored pink are considered "not significant".  '''''How many transcription factors are green or "significant"?'''''
 
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#* '''''Copy the table of results from the web page and paste it into a new Excel workbook to preserve the results.'''''
 
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#** '''''Upload the Excel file to OWW or Box and link to it in your electronic lab notebook.'''''
 
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#** '''''Are GLN3 or HAP4 on the list?  If so, what is their "% in user set", "% in YEASTRACT", and "p value".'''''
 
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# For the mathematical model that we will build, we need to define a ''gene regulatory network'' of transcription factors that regulate other transcription factors.  We can use YEASTRACT to assist us with creating the network.  We want to generate a network with approximately 15-30 transcription factors in it. 
 
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#* You need to select from this list of "significant" transcription factors, which ones you will use to run the model.  You will use these transcription factors and add GLN3 and HAP4 if they are not in your list.  Explain in your electronic notebook how you decided on which transcription factors to include.  Record the list and your justification in your electronic lab notebook.
 
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#* Go back to the YEASTRACT database and follow the link to ''[http://www.yeastract.com/formgenerateregulationmatrix.php Generate Regulation Matrix]''.
 
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#* Copy and paste the list of transcription factors you identified (plus HAP4 and GLN3) into both the "Transcription factors" field and the "Target ORF/Genes" field.
 
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#* We are going to use the "Regulations Filter" options of "Documented", "'''Only''' DNA binding evidence"
 
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#** Click the "Generate" button.
 
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#** In the results window that appears, click on the link to the "Regulation matrix (Semicolon Separated Values (CSV) file)" that appears and save it to your Desktop.  Rename this file with a meaningful name so that you can distinguish it from the other files you will generate.
 
  
 
==Acknowledgements==
 
==Acknowledgements==
  
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#Met outside of class and worked in class with [[User:Kwrigh35|Katie Wright]] to discuss any questions we had throughout the process of completing the Week 10 assignment.
+
#Worked in class with [[User:Kwrigh35|Katie Wright]] to discuss any questions we had throughout the process of completing the Week 10 assignment.
 +
#Recieved help from both [[User:Dondi|Dondi]] and [[User:Kdahlquist|Dr. Dahlquist]] during the allotted class period.
 +
#Instructions in electronic notebook were copied and modified from [[Week 10|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.'''
 
'''While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.'''
Line 117: Line 112:
 
==References==
 
==References==
  
−
#LMU BioDB 2017. (2017). Week 10. Retrieved October 31, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_10
+
# LMU BioDB 2017. (2017). Week 10. Retrieved October 31, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_10
 +
# Short Time-series Expression Miner (STEM). (2006). Retrieved November 29, 2017, from http://www.cs.cmu.edu/~jernst/stem/
 +
# Gene Ontology Consortium. (2017). The Gene Ontology. Retrieved November 29, 2017, from http://geneontology.org
 +
# Glucose 6-phosphate metabolic process. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0000054
 +
# Cytoskeletal protein binding. The Gene Ontology. Retrieved November 29, 2017, http://amigo.geneontology.org/amigo/term/GO:0008092
 +
# Detoxification. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0098754
 +
# Actin binding. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0003779
 +
# Mitochondrion. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0005739
 +
# Whole membrane. The Gene Ontology. Retrieved November 29, 2017, from http://amigo.geneontology.org/amigo/term/GO:0098805
  
 
[[Category:Journal Entry]]
 
[[Category:Journal Entry]]

Latest revision as of 04:49, 30 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. 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
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