This journal entry is due on Tuesday, April 14 at midnight PDT (Monday night/Tuesday morning). NOTE that the server records the time as Eastern Daylight Time (EDT). Therefore, midnight will register as 03:00.
Individual Journal Assignment
- Store this journal entry as "username Week 12" (i.e., this is the text to place between the square brackets when you link to this page).
- Create the following set of links. (HINT: These links should all be in your personal template that you created for the Week 1 Assignment; you should then simply invoke your template on each new journal entry.)
- Link to your journal entry from your user page.
- Link back from your journal entry to your user page.
- Link to this assignment from your journal entry.
- Don't forget to add the "BIOL398-04/S15" category to the end of your wiki page.
For your assignment this week, you will keep an electronic laboratory notebook on your individual wiki page that records all the manipulations you perform on the data and the answers to the questions throughout the protocol. We will be working on the protocols in class on Thursday, April 9. Whatever you do not finish in class will be homework to be completed by the Week 12 journal deadline.
REMINDER: you should "turn on" the file extensions using the instructions found on the Help page before beginning today's work.
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. We will explore this using the YEASTRACT database.
- Open the gene list in Excel for the profile/cluster that you analyzed for the Week 11 Assignment.
- Copy the list of gene IDs onto your clipboard.
- Launch a web browser and go to the YEASTRACT database.
- On the left panel of the window, click on the link to Rank by TF.
- Paste your list of genes from your cluster into the box labeled ORFs/Genes.
- Check the box for Check for all TFs.
- Accept the defaults for the Regulations Filter (Documented, DNA binding plus expression evidence)
- Do not apply a filter for "Filter Documented Regulations by environmental condition".
- Rank genes by TF using: The % of genes in the list and in YEASTRACT regulated by each TF.
- Click the Search button.
- Answer 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"?
- List the "significant" transcription factors on your wiki page, along with the corresponding "% in user set", "% in YEASTRACT", and "p value".
- Are CIN5, GLN3, HMO1, and ZAP1 on the list?
- For the mathematical model that we will build in class, 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 and your partner will need to analyze the same gene regulatory network for your modeling project. Compare the lists of "significant" factors that you and your partner generated. How many of the transcription factors appear in both of your lists? You will use these transcription factors and add CIN5, GLN3, HMO1, and ZAP1 if they are not in your list. If the overlap in the lists between you and your partner does not add up to the 15-30 factors required, use your discretion to add transcription factors from either of your lists (the non-overlapping ones) until you reach a list of 15-30 factors. 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.
- Go back to the YEASTRACT database and follow the link to Generate Regulation Matrix.
- Copy and paste the list of transcription factors you identified (plus CIN5, GLN3, HMO1, and ZAP1) into both the "Transcription factors" field and the "Target ORF/Genes" field.
- We are going to generate several regulation matrices, with different "Regulations Filter" options.
- For the first one, accept the defaults: "Documented", "DNA binding plus expression evidence"
- Click the "Generate" button.
- 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.
- Repeat these steps to generate a second regulation matrix, this time applying the Regulations Filter "Documented", "Only DNA binding evidence".
- Repeat these steps a third time to generate a third regulation matrix, this time applying the Regulations Filter "Documented", DNA binding and expression evidence".
Analyzing and Visualizing Your Gene Regulatory Networks
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.
- 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".
- Now we will look at some of the network properties. Again, repeat these steps for each of the three gene regulatory matrices you generated above. See this file for an example of how to do the following instructions.
- Create a new worksheet and call it "degree". Copy and paste your adjacency matrix from the "network" sheet into this new worksheet.
- In the first empty cell in column A, type "Out-degree". In the cell to the right of that in Column B, type the equation
=SUM( and select the range of cells in column B that has 1's and 0's in it, close the parentheses, and press Enter. This quantity is the number of genes that the transcription factor in that column is controlling, or the out-degree. Copy and paste that equation across all of the columns.
- In Cell 1 of the first empty column to the right of the adjacency matrix, type "In-degree". In Cell 2 of this column, type the equation
=SUM( and select the entire row of 1's and 0's, close the parentheses, and press Enter. This quantity is the number of transcription factors that regulate the gene in that row, or the in-degree. Copy and paste the equation down the entire column, including the row that contains the out-degree sums.
- The number in the lower right-hand corner, the sum of sums, is the total number of edges in the adjacency matrix. We would like to see about 50 (40-60 or so) edges in the matrix. If the matrix is too dense, it will slow down the modeling program because it will be difficult to estimate the parameters in the model.
- We want to plot the degree distributions for each of your gene regulatory networks. In the "degree" worksheet, create three columns to the right called "Frequency", "In-degree total", and "Out-degree total". In the "Frequency" column, number sequentially from 1 to the largest degree number in your calculations above. In the "In-degree total" column, type the number of genes with that in-degree for each of the frequencies. In the "Out-degree total" column, type the number of genes with that out-degree for each of the frequencies.
- Select the "Frequency", "In-degree total", and "Out-degree total" columns. Go to the "Insert" tab and select the column chart type to insert a plot of the degree distribution. Copy and paste the charts for each gene regulatory matrix into your PowerPoint presentation.
- Now we will visualize what these gene regulatory networks look like with the GRNsight software.
- Go to the GRNsight home page (you can either use the version on the home page or the beta version, which has slightly different visualization properties).
- Select the menu item File > Open and select one of 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. Repeat with the other two regulation matrix files. You will want to arrange the genes in the same order for each screenshot so that the graphs can be easily compared.
- Write a paragraph discussing and explaining the results of each aspect of today's work.
- Determining candidate transcription factors that regulate a cluster of genes from your dataset.
- Creating three candidate gene regulatory networks.
- Determining the total number of edges and degree distribution of your three gene regulatory networks.
- Visualizing the networks.
- Choosing a particular gene regulatory network to pursue for the modeling.
Corrections to Week 12 Individual Journal Assignment
Please go back and correct your Week 12 individual journal entries based on the feedback you received in class on Thursday, April 16. The corrections are due by the Week 13 deadline of midnight Tuesday, April 21 PDT. Grades will be not be assigned for the Week 12 entries until after the correction deadline. Make sure your Week 12 journal entry has the following:
- You and your partner should have the same network with the same genes.
- Only delete genes from the network that have zeroes in both the column and row for that gene.
- Give the number of edges and degree distribution charts for the small (binding and expression), medium (binding only), and large (binding plus expression) networks.
- Show the GRNsight graph for the small, medium, and large networks in your PowerPoint presentation.
- Use only your medium network for the Week 13 protocol. You and your partner should have identical input spreadsheets after following this procedure. You should each do the procedure independently, however, so that you can check each other's work.
- Store your shared journal entry in the shared Class Journal Week 12 page. If this page does not exist yet, go ahead and create it (congratulations on getting in first :) )
- Link to your journal entry from your user page.
- Link back from the journal entry to your user page.
- Sign your portion of the journal with the standard wiki signature shortcut (
- Add the "BIOL398-04/S15" category to the end of the wiki page (if someone has not already done so).
- What aspect of this assignment came most easily to you?
- What aspect of this assignment was the most challenging for you?
- What (yet) do you not understand?