Difference between revisions of "Emmatyrnauer Week 14"

Revision as of 23:51, 30 November 2017

Electronic notebook for the continuation of Week 10 Data Analysis

Using YEASTRACT to Infer which Transcription Factors Regulate a Cluster of Genes

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. I explored this using the YEASTRACT database.

1. I opened the gene list in Excel for the one of the significant profiles from my stem analysis (profile 45). I chose this cluster because it has a clear cold shock/recovery up/down pattern. It is also a large cluster.
   • I copied the list of gene IDs from my clipboard (C6:C585).
2. I launched a web browser and went to the YEASTRACT database.
   • On the left panel of the window, I clicked on the link to Rank by TF.
   • I pasted the list of genes from the cluster (profile 45) into the box labeled ORFs/Genes.
   • I checked the box for Check for all TFs.
   • I accepted the defaults for the Regulations Filter (Documented, DNA binding plus expression evidence)
   • I did not apply a filter for "Filter Documented Regulations by environmental condition".
   • I ranked genes by TF using: The % of genes in the list and in YEASTRACT regulated by each TF.
   • I clicked the Search button.
3. I answered the following questions:
   • 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"?
     • 23 transcription factors are green/significant
   • I copied the table of results from the web page and pasted it into a new Excel workbook to preserve the results.
     • I uploaded the Excel file to OWW or Box and linked to it in my electronic lab notebook.
     • Is your transcription factor on the list? If so, what is their "% in user set", "% in YEASTRACT", and "p value". (Note that this doesn't apply to the wt strain).
4. For the mathematical model and GRNsight, 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.
   • 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.
   • I went back to the YEASTRACT database and followed the link to Generate Regulation Matrix.
   • I copied and pasted the list of transcription factors I identified into both the "Transcription factors" field and the "Target ORF/Genes" field. I had to delete the spaces before each of the names for the regulatory network to generate properly.
   • I used the "Regulations Filter" options of "Documented", "Only DNA binding evidence"
     • I clicked the "Generate" button.
     • In the results window that appeared, I clicked on the link to the "Regulation matrix (Semicolon Separated Values (CSV) file)" that appeared and saved it to your Desktop. I renamed this file to "RegulationMatrix_profile45significant_YEASTRACT_emmat"

Visualizing Your Gene Regulatory Networks with GRNsight

We will analyze the regulatory matrix files you generated above in Microsoft Excel and visualize them using GRNsight to determine which one will be appropriate to pursue further in the modeling.

1. First we need to properly format the output files from YEASTRACT. You will repeat these steps for each of the three files you generated above.
   • Open the file in Excel. It will not open properly in Excel because a semicolon was used as the column delimiter instead of a comma. To fix this, Select the entire Column A. Then go to the "Data" tab and select "Text to columns". In the Wizard that appears, select "Delimited" and click "Next". In the next window, select "Semicolon", and click "Next". In the next window, leave the data format at "General", and click "Finish". This should now look like a table with the names of the transcription factors across the top and down the first column and all of the zeros and ones distributed throughout the rows and columns. This is called an "adjacency matrix." If there is a "1" in the cell, that means there is a connection between the trancription factor in that row with that column.
   • Save this file in Microsoft Excel workbook format (.xlsx).
   • Check to see that all of the transcription factors in the matrix are connected to at least one of the other transcription factors by making sure that there is at least one "1" in a row or column for that transcription factor. If a factor is not connected to any other factor, delete its row and column from the matrix. Make sure that you still have somewhere between 15 and 30 transcription factors in your network after this pruning.
     • Only delete the transcription factor if there are all zeros in its column AND all zeros in its row. You may find visualizing the matrix in GRNsight (below) can help you find these easily.
   • For this adjacency matrix to be usable in GRNmap (the modeling software) and GRNsight (the visualization software), we need to transpose the matrix. Insert a new worksheet into your Excel file and name it "network". Go back to the previous sheet and select the entire matrix and copy it. Go to you new worksheet and click on the A1 cell in the upper left. Select "Paste special" from the "Home" tab. In the window that appears, check the box for "Transpose". This will paste your data with the columns transposed to rows and vice versa. This is necessary because we want the transcription factors that are the "regulatORS" across the top and the "regulatEES" along the side.
   • The labels for the genes in the columns and rows need to match. Thus, delete the "p" from each of the gene names in the columns. Adjust the case of the labels to make them all upper case.
   • In cell A1, copy and paste the text "rows genes affected/cols genes controlling".
   • Finally, for ease of working with the adjacency matrix in Excel, we want to alphabatize the gene labels both across the top and side.
     • Select the area of the entire adjacency matrix.
     • Click the Data tab and click the custom sort button.
     • Sort Column A alphabetically, being sure to exclude the header row.
     • Now sort row 1 from left to right, excluding cell A1. In the Custom Sort window, click on the options button and select sort left to right, excluding column 1.
   • Name the worksheet containing your organized adjacency matrix "network" and Save.
2. Now we will visualize what these gene regulatory networks look like with the GRNsight software.
   • Go to the GRNsight home page.
   • Select the menu item File > Open and select the regulation matrix .xlsx file that has the "network" worksheet in it that you formatted above. If the file has been formatted properly, GRNsight should automatically create a graph of your network. Move the nodes (genes) around until you get a layout that you like and take a screenshot of the results. Paste it into your PowerPoint presentation.