Week 10

This journal entry is due on Thursday, November 7, at 12:01 PST. (Monday night/Tuesday morning)

There is an interim deadline for Tuesday, November 5 at 12:01 PST, based on what we do in class.

Overview

• To gain experience with database queries and modeling, the last step in preparation for the final project.
• Everyone will contribute to Creating your team's home page, and in the process, getting yourselves organized for the final project.

Individual Journal Assignment

• Store this journal entry as "username Week 10" (i.e., this is the text to place between the square brackets when you link to this page).
• Invoke your template on your journal entry page so that you:
  • Link from your journal entry page to this Assignment page.
  • Link from your journal entry to your user page.
  • Add the "Journal Entry" category to the end of your wiki page.
• Because you have invoked your template on your user page, you should also have a:
  • Link from your user page to this Assignment page.
  • Link to your journal entry from your user page.
• Note that this week, we will add two new categories, "Group Projects" and a category for your team's name. Please do not add these to your individual templates because we want these categories to be precisely used for the Group Projects and your team, respectively.
• Include both the Acknowledgments and References section as specified by the Week 1 assignment.
• For your assignment this week, electronic laboratory notebook will be modified to fit the assignment that is specific to your role on your team.

Team Membership

For creating the GRNmap Input Workbook, the groups will be the same as for the Week 9 assignment.

The project groups and roles will be announced in class on Tuesday, November 5.

Creating the GRNmap Input Workbook

Now that you have identified the gene regulatory network that you want to model, the next step is to generate the input Excel workbook that you will run in the GRNmap modeling software.

Click here to download a sample workbook on which to base the one specific to your network and microarray data.

Note that when following the instructions below, you need to follow them precisely, to the letter, or GRNmap will return an error.

production_rates sheet

• This sheet contains initial guesses for the production rate parameters, P, for all genes in the network.
• Assuming that the system is in steady state with the relative expression of all genes equal to 1, (P/2) - lambda = 0, where lambda is the degradation rate, is a reasonable initial guess.
• The sheet should contain two columns (from left to right) entitled, "id", "production_rate".
  • The id is an identifier that the user will use to identify a particular gene. In our case, we are using the "StandardName", for example, GLN3.
  • The "production_rate" column should then contain the initial guesses for the P parameter as described above, rounded to four decimal places.
    • The production rates are provided in a Microsoft Access database, which you can download from here.
    • You will perform a query to get the list of production rates for each gene as a group.
    • To perform the query, you will need to follow these steps.
      1. Import a your list of genes to a new table in the database. Click on the "External Data" tab and select the Excel icon with the "up" arrow on it.
      2. Click the "Browse" button and select your Excel file containing your network that you used to upload to GRNsight.
      3. Make sure the button next to "Import the source data into a new table in the current database" and click "OK".
      4. In the next window, select the "network" worksheet, if it hasn't already been automatically selected for you. Click "Next".
      5. In the next window, make sure the "First Row Contains Column Headings" is checked. Click "Next".
      6. In the next window, the left-most column will be highlighted. Change the "Field Name" to "id" if it doesn't say that already. Click "Next".
      7. In the next window, select the button for "Choose my own primary key." and choose the "id" field from the drop down next to it. Click "Next".
      8. In the next field, make sure it says "Import to Table: network". Click Finish.
      9. In the next window you do not need to save the import steps, so just click "Close".
      10. A table called "network" should appear in the list of tables at the left of the window.
      11. Go to the "Create" tab. Click on the icon for "Query Design".
      12. In the window that appears, click on the "network" table and click "Add". Click on the "production_rates" table and click "Add". Click "Close".
      13. The two tables should appear in the main part of the window. We need to tell Access which fields in the two tables correspond to each other. Click on the word "id" in the network table and drag your mouse to the "standard_name" field in the "production_rates" table, and release. You will see a line appear between those two words.
      14. Right-click on the line between those words and select "Join Properties" from the menu that appears. Select Option "2: Include ALL records from 'network' and only those records from 'production_rates' where the joined fields are equal." Click "OK".
      15. Click on the "id" word in the "network" table and drag it to the bottom of the screen to the first column next to the word "Field" and release.
      16. Click on the "production_rate" field in the "production_rates" table and drag it to the bottom of the screen to the second column next to the word "Field" and release.
      17. Right-click anywhere in the gray area near the two tables. In the menu that appears, select "Query Type > Make Table Query...".
      18. In the window that appears, name your table "production_rates_1" because you can't have two tables with the same name in the database. Make sure that "Current Database" is selected and Click "OK".
      19. Go to the "Query Tools: Menus" tab. Click on the exclamation point icon. A window will appear that tells you how many rows you are pasting into a new table. Click "Yes".
      20. Your new "production_rates_1" table will appear in the list at the left. Double-click on that table name to open it.
      21. You can copy the data in this table and paste it back into your Excel workbook. Make sure that when you paste that you use "Paste Special > Paste values" so that the Access formatting doesn't get carried along. You can also choose to export this table to Excel going to the "External Data" tab and selecting the Excel icon with the arrow pointing to the right. Select the workbook you want to export the table to, making sure that "Preserve Access formatting" is not checked. Click "OK", click "Close".
• Note that the genes should be listed in the same order in all the sheets in the Excel workbook.

degradation_rates sheet

• This sheet contains degradation rates for all genes in the network, which are provided by the user.
• Currently, the Dahlquist Lab is using data based on published mRNA half-life data from Neymotin et al. (2006).
  • We converted the half-life data values to the degradation rates by taking the natural log of the half-life and dividing by 2.
• The sheet should contain two columns (from left to right) entitled "id", and "degradation_rate".
  • The id is an identifier that the user will use to identify a particular gene.
  • The "degradation_rate" column should then contain the absolute value of the degradation rate for the corresponding gene as described above, rounded to four decimal places.
    • To obtain these values, you will use the same file, Microsoft Access database that you used to obtain the production rates in the first worksheet. Again, you can copy and paste the values one-by-one or you can follow the instructions to execute a query, substituting the appropriate "degradation_rates" table in the query. Note that you don't need to re-import your "network" table, you just need to create and execute the query.
• Again note, the genes should be listed in the same order in all the sheets in the Excel workbook.

Expression Data Sheets for Individual Yeast Strains

• Expression data can be provided for either a single strain or multiple strains of yeast (for example, the wild type strain and a transcription factor deletion strain).
  • Each strain will have its own sheet in the workbook.
  • Each sheet should be given a unique name that follows the convention "STRAIN_log2_expression", where the word "STRAIN" is replaced by the strain designation, which will appear in the optimization_diagnostics sheet.
    • Everyone in the class will have at least one expression worksheet called "wt_log2_expression".
    • You should have included the transcription factors GLN3, HAP4, and

CIN5 in your network. Thus, we will use the expression data from the dGLN3, dHAP4, dCIN5 deletion strains in our workbooks as well, naming the worksheets "dgln3_log2_expression", "dhap4_log2_expression", and "dcin5_expression".

• • • • If, for some reason, you don't have all three of those genes in your network, only include expression data for the wild type and the genes out of those three that you have in your network.
• The sheet should have the following columns in this order:
  1. "id": list of all genes. The genes should be listed in the same order in all the sheets in the Excel workbook.
  2. The next series of columns should contain the expression data for each gene at a given timepoint given as log2 ratios (log2 fold changes). The column header should be the time at which the data were collected, without any units. For example, the 15 minute timepoint would have a column header "15" and the 30 minute timepoint would have the column header "30". GRNmap supports replicate data for each of the timepoints. Replicate data for the same timepoint should be in columns immediately next to each other and have the same column headers. For example, three replicates of the 15 minute timepoint would have "15", "15", "15" as the column headers.
  3. If data are provided for multiple strains, each strain should have data for the same timepoints, although the number of replicates can vary.
• Include the data for the 15, 30, and 60 minute timepoints, but not the 90 or 120 minute timepoints.
• The data you will be using is contained in the Expression-and-Degradation-rate-database_2019.accdb file that you used to obtain the production and degradation rates.
• It is tedious to copy and paste all of these data by hand, so we will execute a query in Microsoft Access to do it for you. Follow the steps listed for the "production_rates" sheet for each strains expression data. After you import the data into Excel, you will need to change the column headers to "15", "15", etc., as described above.

network sheet

• The network you derived from the YEASTRACT database for the Week 5 assignment can be copied and pasted into this sheet directly. You may need to edit the contents of cell A1, but the rest should be good to go (especially since you previewed it in GRNsight). The description below just explains what is already in this worksheet.
  • This sheet contains an adjacency matrix representation of the gene regulatory network.
  • The columns correspond to the transcription factors and the rows correspond to the target genes controlled by those transcription factors.
  • A “1” means there is an edge connecting them and a “0” means that there is no edge connecting them.
  • The upper-left cell (A1) should contain the text “cols regulators/rows targets”. This text is there as a reminder of the direction of the regulatory relationships specified by the adjacency matrix.
  • The rest of row 1 should contain the names of the transcription factors that are controlling the other genes in the network, one transcription factor name per column.
  • The rest of column A should contain the names of the target genes that are being controlled by the transcription factors heading each of the columns in the matrix, one target gene name per row.
  • The transcription factor names should correspond to the "id" in the other sheets in the workbook. They should be capitalized the same way and occur in the same order along the top and side of the matrix. The matrix needs to be symmetric, i.e., the same transcription factors should appear along the top and left side of the matrix. The genes should be listed in the same order in all the sheets in the Excel workbook.
  • Each cell in the matrix should then contain a zero (0) if there is no regulatory relationship between those two transcription factors, or a one (1) if there is a regulatory relationship between them. Again, the columns correspond to the transcription factors and the rows correspond to the target genes controlled by those transcription factors.

network_weights sheet

• These are the initial guesses for the estimation of the weight parameters, w.
• Since these weights are initial guesses which will be optimized by GRNmap, the content of this sheet can be identical to the "network" sheet.

optimization_parameters sheet

• The optimization_parameters sheet should have two columns (from left to right) entitled, "optimization_parameter" and "value".
• You should copy this worksheet from the sample workbook provided. The only row that you need to modify is row 15, "Strain". Include just the strain designations for which you have a corresponding STRAIN_log2_expression sheet. If you don't have the dgln3, dhap4, or dcin5 expression sheets, then you will delete those from this row. If you do so, make sure that you don't leave any gaps between cells.
• What follows below is an explanation of what the optimization_parameters mean.
  • alpha: Penalty term weighting (from the L-curve analysis)
  • kk_max: Number of times to re-run the optimization loop. In some cases re-starting the optimization loop can improve performance of the estimation.
  • MaxIter: Number of times MATLAB iterates through the optimization scheme. If this is set too low, MATLAB will stop before the parameters are optimized.
  • TolFun: How different two least squares evaluations should be before the program determines that it is not making any improvement
  • MaxFunEval: maximum number of times the program will evaluate the least squares cost
  • TolX: How close successive least squares cost evaluations should be before the program determines that it is not making any improvement.
  • production_function: = Sigmoid (case-insensitive) if sigmoidal model, =MM (case-insensitive) if Michaelis-Menten model
  • L_curve: =0 if an L-curve analysis should NOT be run or =1 if an L-curve analysis SHOULD be run. The L-curve analysis will automatically run sequential rounds of estimation for an array of fixed alpha values (0.8, 0.5, 0.2, 0.1,0.08, 0.05,0.02,0.01, 0.008, 0.005, 0.002, 0.001, 0.0008, 0.0005, 0.0002, and 0.0001). GRNmap makes a copy of the user's selected input workbook and changes alpha to the first alpha in the list. The estimation runs and the resulting parameter values are used as the initial guesses for the next round of estimation with the next alpha value. This process repeats until all alpha values have been run. New input and output workbooks are generated for each alpha value, although currently, the graphs are only saved for the last run.
  • estimate_params =1 if want to estimate parameters and =0 if the user wants to do just one forward run
  • make_graphs =1 to output graphs; =0 to not output graphs
  • fix_P =1 if the user does not want to estimate the production rate, P, parameter, just use the initial guess and never change; =0 to estimate
  • fix_b =1 if the user does not want to estimate the b parameter, just use the initial guess and never change; =0 to estimate
  • expression_timepoints: A row containing a list of the time points when the data was collected experimentally. Should correspond to the timepoint column headers in the STRAIN_log2_expression sheets.
  • Strain: A row containing a list of all of the strains for which there is expression data in the workbook. Should correspond to the "STRAIN" portion of the names of the STRAIN_log2_expression sheets for each strain. Note that GRNmap will run the model for the wild type network (all genes present in the network) and for networks where the gene deleted from the designated STRAIN has been deleted from the network.
  • simulation_timepoints: A row containing a list of the time points at which to evaluate the differential equations to generate the simulated data. This does not need to correspond to the actual measurement times, but should be in the same units (e.g. minutes).

threshold_b sheet

• These are the initial guesses for the estimation of the threshold_b parameters.
• There should be two columns.
  • The left-most column should contain the header "id" and list the standard names for the genes in the model in the same order as in the other sheets.
  • The second column should have the header "threshold_b" and should contain the initial guesses, we are going to use all 0.

Whole Team Journal Assignment: Creating a Team Wiki Page

From this week on, your "Shared Journal Assignments" will become "Team Journal Assignments". For this week, some preliminary tasks are assigned to your team to kickstart your final projects.

1. Name your team and create your team home page on the wiki.
   • The name of your team home page should simply be the team name.
   • This page will be the main place from which your team project will be managed. Include all of the information/links that you think will be useful for your team to organize your work and communicate with each other and with the instructors. Hint: the kinds of things that are on your own User pages and on the course Main page can be used as a guide.
2. Create a link to your team's page on the course Main page.
3. Create a template for your team with useful information and links that you will invoke on all pages that you will create for the project.
   • Create a category using your team name and include it on your team's template so that it also gets used on all pages you will create for the project. Also use include the category "Group Projects" in your template.
     • However, please do not add these categories to your own individual templates because we want them to precisely mark pages having to do with the Group Projects and your team, respectively.
4. Each person needs to write a short executive summary of that person's progress on the project for the week, with links to the relevant individual journal pages (which will have more detailed information).
5. Each team member should reflect on the team's progress:
   1. What worked?
   2. What didn't work?
   3. What will I do next to fix what didn't work?
6. Note that you will be directed to add specific information to your team's pages in the individual portion of the assignment for this and future weeks.