BIOL478/S15:Microarray Data Analysis

Laboratory 10: Microarray Data Analysis Wednesday, April 22, Monday, April 27, and Wednesday, April 29

Before We Begin

Viewing File Extensions

• The Windows 7 operating systems defaults to hiding file extensions. To turn them back on, do the following:

  

  Folder Options window

  1. Go to the Start menu and select "Control Panel".
  2. In the window that appears, search for "Folder Options" in the search field in the upper right hand corner.
  3. Click on "Folder Options" in the main window.
  4. When the Folder Options window appears, click on the View tab.
  5. Uncheck the box for "Hide extensions for known file types".
  6. Click the OK button.
• The computers in Seaver 120 are are set to erase all custom user settings and restore the defaults once they have been restarted, so you will probably have to do this each time you sit down to using these computers.

Background

This is a list of steps required to analyze DNA microarray data.

1. Quantitate the fluorescence signal in each spot
2. Calculate the ratio of red/green fluorescence
3. Log₂ transform the ratios
4. Normalize the ratios on each microarray slide
5. Normalize the ratios for a set of slides in an experiment
   • Steps 4-5 were performed for you by instructor using a script written in the R statistical programming language
   • You will perform the following steps:
6. Perform statistical analysis on the ratios
7. Compare individual genes with known data
   • Steps 6-7 are performed in Microsoft Excel
8. Pattern finding algorithms (clustering)
   • We will use software called STEM for clustering
9. Maping onto biological pathways
   • We will use software called GenMAPP for mapping onto biological pathways
10. Identifying regulatory transcription factors responsible for observed changes in gene expression

We will combine the data that you obtained in the Δhap4 cold shock and recovery experiment for flasks 3 and 4 with data obtained in my research lab for flask 2 (flask 1 hasn't been hybridized yet). You will also be analyzing the wild type data obtained in my research lab for comparison with the deletion strain.

You will download an Excel spreadsheet named "BIOL478_wt_dHAP4-F2-F3-F4_compiled_normalized_data_20150422.xlsx" that contains the normalized log fold changes from MyLMUConnect for use in the following protocol.

Open a Microsoft Word document to use as your electronic lab notebook for the three computer lab sessions. Take notes as you go along to record the specific manipulations you perform on the data. Record in this document the answers to any questions stated in the protocol. You will need this information for your final laboratory report.

Experimental Design

• The Excel Workbook contains two worksheets called "wt_compiled_normalized_data" and "dHAP4_compiled_normalized_data", for the wild type and dHAP4 data, respectively. Each of the worksheets is organized as follows:
• Each row contains the data for one gene (one spot on the microarray).
• The first column (labeled "MasterIndex") numbers the rows in the spreadsheet so that we can sort the data back into the same order later.
• The second column (labeled "ID") contains the gene identifier from the Saccharomyces Genome Database.
• The third column (labeled "StandardName") contains the Standard Name for each gene from the Saccharomyces Genome Database.
• Each subsequent column contains the normalized log₂ ratio of the red/green fluorescence from each microarray hybridized in the experiment (steps 1-5 above having been done for you by the scanner software and R).
• Each of the column headings from the data begin with the experiment name ("wt" for wild type or "dHAP4" for the Δhap4 data). "LogFC" stands for "Log₂ Fold Change" which is the Log₂ red/green ratio. The timepoints are designated as "t" followed by a number in minutes. Replicates are numbered as "-1", "-2", etc. after the timepoint, indicating which flask the cells were harvested from.
• The timepoints are t15, t30, t60 (cold shock at 13°C) and t90 and t120 (cold shock at 13°C followed by 30 or 60 minutes of recovery at 30°C).
• For the dHAP4 strain, there are three replicates for each timepoint (referring to the Flasks 2, 3, and 4).
• Record the number of replicates for each timepoint for the wild type strain in your electronic lab notebook.
• In the following protocol, one lab partner will crunch the wild type data and one partner will crunch the dHAP4 data. You will then combine your results for further analysis and interpretation.

Perform statistical analysis on the ratios

Statistical Analysis Part 1: ANOVA

One partner will follow this protocol for the wild type strain and one partner will follow this protocol for the dHAP4 strain.

1. Create a new worksheet, naming it either "wt_ANOVA" or "dHAP4_ANOVA" as appropriate.
2. Copy all of the data from the "compiled_normalized_data" sheet for your strain into your new worksheet.
3. At the top of the first column to the right of your data, create five column headers of the form (STRAIN)_AvgLogFC_(TIME) where (STRAIN) is wt or dHAP4 and (TIME) is 15, 30, etc.
4. In the cell below the (STRAIN)_AvgLogFC_t15 header, type =AVERAGE(
5. Then highlight all the data in row 2 associated with (STRAIN) and t15, press the closing paren key (shift 0),and press the "enter" key.
6. This cell now contains the average of the log fold change data from the first gene at t=15 minutes.
7. Click on this cell and position your cursor at the bottom right corner. You should see your cursor change to a thin black plus sign (not a chubby white one). When it does, double click, and the formula will magically be copied to the entire column of 6188 other genes.
8. Repeat steps (4) through (7) with the t30, t60, t90, and the t120 data.
9. Now in the first empty column to the right of the (STRAIN)_AvgLogFC_t120 calculation, create the column header (STRAIN)_ss_HO.
10. In the first cell below this header, type =SUMSQ(
11. Highlight all the data in row 2 for your (STRAIN), press the closing paren key (shift 0),and press the "enter" key.
    • The data highlighted here will be same as in (10).
12. In the next empty column to the right of (STRAIN)_ss_HO, create the column headers (STRAIN)_ss_(TIME) as in (3).
13. Make a note of how many data points you have at each time point for your strain. For dHAP4, it will be "3", but for the wild type it will be "4" or "5". Count carefully. Also, make a note of the total number of data points. For dHAP4, this number will be 15, but for wt it should be 23 (double-check).
14. In the first cell below the header (STRAIN)_ss_t15, type =SUMSQ(<range of cells for logFC_t15>)-<number of data points>*<AvgLogFC_t15>^2 and hit enter.
    • The phrase <range of cells for logFC_t15> should be replaced by the data range associated with t15.
    • The phrase <number of data points> should be replaced by the number of data points for that timepoint (either 3, 4, or 5).
    • The phrase <AvgLogFC_t15> should be replaced by the cell number in which you computed the AvgLogFC for t15, and the "^2" squares that value.
    • Upon completion of this single computation, use the Step (7) trick to copy the formula throughout the column.
15. Repeat this computation for the t30 through t120 data points. Again, be sure to get the data for each time point, type the right number of data points, and get the average from the appropriate cell for each time point, and copy the formula to the whole column for each computation.
16. In the first column to the right of (STRAIN)_ss_t120, create the column header (STRAIN)_SS_full.
17. In the first row below this header, type =sum(<range of cells containing "ss" for each timepoint>) and hit enter.
18. In the next two columns to the right, create the headers (STRAIN)_Fstat and (STRAIN)_p-value.
19. Recall the number of data points from (13): call that total n.
20. In the first cell of the (STRAIN)_Fstat column, type =((n-5)/5)*(<(STRAIN)_ss_HO>-<(STRAIN)_SS_full>)/<(STRAIN)_SS_full> and hit enter.
    • Don't actually type the n but instead use the number from (13).
    • Replace the phrase (STRAIN)_ss_HO with the cell designation.
    • Replace the phrase <(STRAIN)_SS_full> with the cell designation.
    • Copy to the whole column.
21. In the first cell below the (STRAIN)_p-value header, type =FDIST(<(STRAIN)_Fstat>,5,n-5) replacing the phrase <(STRAIN)_Fstat> with the cell designation and the "n" as in (13) with the number of data points total. Copy to the whole column.
22. Before we move on to the next step, we will perform a quick sanity check to see if we did all of these computations correctly.
    • Click on cell A1 and click on the Data tab. Select the Filter icon (looks like a funnel). Little drop-down arrows should appear at the top of each column. This will enable us to filter the data according to criteria we set.
    • Click on the drop-down arrow on your (STRAIN)_p-value column. Select "Number Filters". In the window that appears, set a criterion that will filter your data so that the P value has to be less than 0.05.
    • Excel will now only display the rows that correspond to data meeting that filtering criterion. A number will appear in the lower left hand corner of the window giving you the number of rows that meet that criterion. We will check our results with the rest of the class to make sure that the computations were performed correctly.

Calculate the Bonferroni and p value Correction

1. Now we will perform adjustments to the p value to correct for the multiple testing problem. Label the next two columns to the right with the same label, (STRAIN)_Bonferroni_p-value.
2. Type the equation =<(STRAIN)_p-value>*6189, Upon completion of this single computation, use the Step (10) trick to copy the formula throughout the column.
3. Replace any corrected p value that is greater than 1 by the number 1 by typing the following formula into the first cell below the second (STRAIN)_Bonferroni_p-value header: =IF(r2>1,1,r2). Use the Step (10) trick to copy the formula throughout the column.

Calculate the Benjamini & Hochberg p value Correction

1. Insert a new worksheet named "(STRAIN)_B&H".
2. Copy and paste the "MasterIndex" and "ID" columns from your previous worksheet into the first two columns of the new worksheet.
3. For the following, use Paste special > Paste values. Copy your unadjusted p values from your ANOVA worksheet and paste it into Column C.
4. Select all of columns A, B, and C. Sort by ascending values on Column C. Click the sort button from A to Z on the toolbar, in the window that appears, sort by column C, smallest to largest.
5. Type the header "Rank" in cell D1. We will create a series of numbers in ascending order from 1 to 6189 in this column. This is the p value rank, smallest to largest. Type "1" into cell D2 and "2" into cell D3. Select both cells A2 and A3. Double-click on the plus sign on the lower right-hand corner of your selection to fill the column with a series of numbers from 1 to 6189.
6. Now you can calculate the Benjamini and Hochberg p value correction. Type (STRAIN)_B-H_p-value in cell E1. Type the following formula in cell E2: =(C2*6189)/D2 and press enter. Copy that equation to the entire column.
7. Type "STRAIN_B-H_p-value" into cell F1.
8. Type the following formula into cell F2: =IF(E2>1,1,E2) and press enter. Copy that equation to the entire column.
9. Select columns A through F. Now sort them by your MasterIndex in Column A in ascending order.
10. Copy column F and use Paste special > Paste values to paste it into the next column on the right of your ANOVA sheet.

• Upload the .xlsx file that you have just created to LionShare. Send Dr. Dahlquist an e-mail with the link to the file (e-mail kdahlquist at lmu dot edu).

Sanity Check: Number of genes significantly changed

Before we move on to further analysis of the data, we want to perform a more extensive sanity check to make sure that we performed our data analysis correctly. We are going to find out the number of genes that are significantly changed at various p value cut-offs.

• Go to your (STRAIN)_ANOVA worksheet.
• Select row 1 (the row with your column headers) and select the menu item Data > Filter > Autofilter (The funnel icon on the Data tab). Little drop-down arrows should appear at the top of each column. This will enable us to filter the data according to criteria we set.
• Click on the drop-down arrow for the unadjusted p value. Set a criterion that will filter your data so that the p value has to be less than 0.05.
  • How many genes have p < 0.05? and what is the percentage (out of 6189)?
  • How many genes have p < 0.01? and what is the percentage (out of 6189)?
  • How many genes have p < 0.001? and what is the percentage (out of 6189)?
  • How many genes have p < 0.0001? and what is the percentage (out of 6189)?
• When we use a p value cut-off of p < 0.05, what we are saying is that you would have seen a gene expression change that deviates this far from zero by chance less than 5% of the time.
• We have just performed 6189 hypothesis tests. Another way to state what we are seeing with p < 0.05 is that we would expect to see this a gene expression change for at least one of the timepoints by chance in about 5% of our tests, or 309 times. Since we have more than 309 genes that pass this cut off, we know that some genes are significantly changed. However, we don't know which ones. To apply a more stringent criterion to our p values, we performed the Bonferroni and Benjamini and Hochberg corrections to these unadjusted p values. The Bonferroni correction is very stringent. The Benjamini-Hochberg correction is less stringent. To see this relationship, filter your data to determine the following:
  • How many genes are p < 0.05 for the Bonferroni-corrected p value? and what is the percentage (out of 6189)?
  • How many genes are p < 0.05 for the Benjamini and Hochberg-corrected p value? and what is the percentage (out of 6189)?
• In summary, the p value cut-off should not be thought of as some magical number at which data becomes "significant". Instead, it is a moveable confidence level. If we want to be very confident of our data, use a small p value cut-off. If we are OK with being less confident about a gene expression change and want to include more genes in our analysis, we can use a larger p value cut-off.
• Comparing results with known data: the expression of the gene NSR1 (ID: YGR159C)is known to be induced by cold shock. Find NSR1 in your dataset. What is its unadjusted, Bonferroni-corrected, and B-H-corrected p values? What is its average Log fold change at each of the timepoints in the experiment? Note that the average Log fold change is what we called "STRAIN)_AvgLogFC_(TIME)" in step 3 of the ANOVA analysis.
• You and your partner should compare the numbers you got between the wild type strain and the dHAP4. You will be reporting this information in your final paper in the course, organized as a table. Use this sample PowerPoint slide to see how your table should be formatted.

Clustering and Gene Ontology Analysis with STEM

1. Prepare your microarray data file for loading into STEM.
   • Insert a new worksheet into your Excel workbook, and name it "(STRAIN)_stem".
   • Select all of the data from your "(STRAIN)_ANOVA" worksheet and Paste special > paste values into your "(STRAIN)_stem" worksheet.
     • Your leftmost column should have the column header "MasterIndex". Rename this column to "SPOT". Column B should be named "ID". Rename this column to "Gene Symbol". Delete the column named "StandardName".
     • Filter the data on the B-H corrected p value to be > 0.05 (that's greater than in this case).
       • 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.
     • Delete all of the data columns EXCEPT for the Average Log Fold change columns for each timepoint (for example, wt_AvgLogFC_t15, etc.).
     • Rename the data columns with just the time and units (for example, 15m, 30m, etc.).
     • 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.
       • Note that you should turn on the file extensions if you have not already done so.
2. Now download and extract the STEM software. Click here to go to the STEM web site.
   • Click on the download link, register, and download the stem.zip file to your Desktop.
   • Unzip the file. In Seaver 120, you can right click on the file icon and select the menu item 7-zip > Extract Here.
   • This will create a folder called stem. Inside the folder, double-click on the stem.cmd or the stem.jar to launch the STEM program.
3. Running STEM
   1. 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.
      • Click on the radio button No normalization/add 0.
      • Check the box next to Spot IDs included in the data file.
   2. 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.
   3. 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.
   4. In section 4 (Execute) click on the yellow Execute button to run STEM.
4. Viewing and Saving STEM Results
   1. 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.
      • 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.
      • Take 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 paste it into a PowerPoint presentation to save your figures.
   2. 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.
      • Take a screenshot of each of the individual profile windows and save the images in your 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.
        • Upload these files to LionShare and e-mail a link to Dr. Dahlquist. (It will be easier to zip all the files together and upload them as one file).
      • 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!
        • Upload these files to LionShare and e-mail a link to Dr. Dahlquist. (It will be easier to zip all the files together and upload them as one file).
5. Analyzing and Interpreting STEM Results
   1. 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 early (first three) timepoints. You and your partner will choose the same profile so that you can compare your results between the two strains. Answer the following:
      • Why did you select this profile? In other words, why was it interesting to you?
      • How many genes belong to this profile?
      • How many genes were expected to belong to this profile?
      • 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.
      • 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?
      • Select 10 Gene Ontology terms from your filtered list (either p < 0.05 or corrected p < 0.05). Look up the definitions for each of the terms at http://geneontology.org. For your final lab report, 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?
        • To easily look up the definitions, go to http://geneontology.org.
        • Copy and paste the GO ID (e.g. GO:0044848) into the search field at the upper left of the page called "Search GO Data".
        • In the results page, click on the button that says "Link to detailed information about <term>, in this case "biological phase"".
        • The definition will be on the next results page, e.g. here.

Files to Upload to LionShare from Day 1

• Excel spreadsheet with all calculations
• tab-delimited text file used as input for STEM
• PowerPoint file containing screenshots of STEM results
• Profile gene table file from the profile you have selected to analyze
• Profile GO table file from the profile you have selected to analyze

• You can zip these files together by putting them in a folder, right-clicking on the folder icon and choosing 7-zip > Add to archive... In the window that opens, make sure to select "zip" for the archive format.
• Please upload your zipped file to LionShare and e-mail me the link to the file.

Statistical Analysis Part 2: Modified T Test

Last week we performed an ANOVA to determine if any genes had a gene expression change that was significantly different than zero at any timepoint. Now we will perform a modified t test to determine if any genes had a gene expression change that was significantly different than zero at each timepoint. You will perform your analysis on the same strain that you did last week, adding these calculations to the same Excel workbook.

• Insert a new worksheet into your Excel workbook and name it either "wt_ttest" or "dHAP4_ttest".
• Go back to the "compiled_normalized_data" worksheet for your strain, Select All and Copy. Go to your new "_ttest" worksheet, click on the upper, left-hand cell (cell A1) and Select "Paste Special" from the Edit menu. A window will open: click on the radio button for "Values" and click OK. This will paste the numerical result into your new worksheet instead of the equation which must make calculations on the fly.
  • There may be some non-numerical values in some of the cells in your worksheet. This is due to errors created when Excel tries to compute an equation on a cell that has no data. We need to go through and remove these error messages before going on to the next step.
  • Scan through your spreadsheet to find an example of the error message. Then go to the Edit menu and Select Replace. A window will open, type the text you are replacing in the "Find what:" field. In the "Replace with:" field, enter a single space character. Click on the button "Replace All" and record the number of replacements in your lab notebook.
• Go to the empty columns to the right on your worksheet. Create new column headings in the top cells to label the average log fold changes that you will compute. Name them with the pattern <dHAP4>_<AvgLogFC>_<tx> where you use the appropriate text within the <> and where x is the time. For example, "dHAP4_AvgLogFC_t15".
• Compute the average log fold change for the replicates for each timepoint by typing the equation:

=AVERAGE(range of cells in the row for that timepoint)

into the second cell below the column heading. For example, your equation might read

=AVERAGE(C2:F2)

Copy this equation and paste it into the rest of the column.

• Create the equation for the rest of the timepoints and paste it into their respective columns. Note that you can save yourself some time by completing the first equation for all of the averages and then copy and paste all the columns at once.
• Go to the empty columns to the right on your worksheet. Create new column headings in the top cells to label the T statistic that you will compute. Name them with the pattern <dHAP4>_<Tstat>_<tx> where you use the appropriate text within the <> and where x is the time. For example, "dHAP4_Tstat_t15". You will now compute a T statistic that tells you whether the normalized average log fold change is significantly different than 0 (no change in expression). Enter the equation into the second cell below the column heading:

=AVERAGE(range of cells)/(STDEV(range of cells)/SQRT(number of replicates))

For example, your equation might read:

=AVERAGE(C2:F2)/(STDEV(C2:F2)/SQRT(4))

(NOTE: in this case the number of replicates is 4. Be careful that you are using the correct number of parentheses.) Copy the equation and paste it into all rows in that column. Create the equation for the rest of the timepoints and paste it into their respective columns. Note that you can save yourself some time by completing the first equation for all of the T statistics and then copy and paste all the columns at once.

• Go to the empty columns to the right on your worksheet. Create new column headings in the top cells to label the P value that you will compute. Name them with the pattern <dHAP4>_<Pval>_<tx> where you use the appropriate text within the <> and where x is the time. For example, "dHAP4_Pval_t15". In the cell below the label, enter the equation:

=TDIST(ABS(cell containing T statistic),degrees of freedom,2)

For example, your equation might read:

=TDIST(ABS(AE2),3,2)

The number of degrees of freedom is the number of replicates minus one. Copy the equation and paste it into all rows in that column.

• As with the ANOVA, we encounter the multiple testing problem here as well. We could perform the Bonferroni and/or Benjamini & Hochberg corrections on these p values, but we won't in the interests of time.
• We will, however, perform the "sanity check" as follows:
  • Determine how many genes have a p value < 0.05 at each timepoint.
  • Keeping the "Pval" filter at p < 0.05, How many have an average log fold change of > 0.25 and p < 0.05 at each timepoint? How many have an average log fold change of < -0.25 and p < 0.05 at each timepoint? (These log fold change cut-offs represent about a 20% fold change in expression.)
  • Arrange these data in a table for your final report.
• Insert a new worksheet and name it "wt_final" or "dHAP4_final".
• Go back to the "ANOVA" worksheet for your strain and Select All and Copy.
• Go to your new sheet and click on cell A1 and select Paste Special, click on the Values radio button, and click OK. This is your final worksheet that compiles the statistical analysis for your strain.
  • Delete the columns containing the "ss" calculations, just retaining the individual log fold change data, the average log fold change data, the Fstat and p value. For the Bonferroni and B&H p values, just keep one column where we replaced all values > 1 with 1.
• Now go to your "_ttest" worksheet. Copy just the columns containing the Fstats and P values for the individual timepoints and Paste special > Paste values into your "final" to the right of the previous data.
• Select all of the columns containing Fold Changes. Select the menu item Format > Cells. Under the number tab, select 2 decimal places. Click OK.
• Select all of the columns containing T statistics or P values. Select the menu item Format > Cells. Under the number tab, select 4 decimal places. Click OK.
• We will now format this file for use with GenMAPP.
  • Currently, the "MasterIndex" column is the first column in the worksheet. We need the "ID" column to be the first column. Select Column B and Cut. Right-click on Cell A1 and select "Insert cut cells". This will reverse the position of the columns.
  • Insert a new empty column in Column B. Type "SystemCode" in the first cell and "D" in the second cell of this column. Use our trick to fill this entire column with "D".
• Upload the .xlsx file that you have just created to LionShare. Check the box to overwrite your previous file of the same filename to update your previous file with the changes. Send an e-mail to your partner with the link to the file because you will both need to now merge your data for the next step.
  • When you get your partner's file, you will copy all of the data from their "_final" worksheet and paste it into the columns to the right of your strain's data. Once you have determined that your data is lined up properly, you can delete the second set of columns that contain the ID, SystemCode, MasterIndex, and StandardName.
  • Make sure to save this work as your .xlsx file. Now save this worksheet as a tab-delimited text file for use with GenMAPP in the next section.

Map onto Biological Pathways using GenMAPP

Each time you launch GenMAPP, you need to make sure that the correct Gene Database (.gdb) is loaded.

• Look in the lower left-hand corner of the window to see which Gene Database has been selected.
• If you need to change the Gene Database, select Data > Choose Gene Database. Navigate to the directory C:\GenMAPP 2 Data\Gene Databases and choose the correct one for your species.
• For the exercise today, if the yeast Gene Database is not present on your computer, you will need to download it. Click this link to download the yeast Gene Database.
• Unzip the file and save it, Sc-Std_20060526.gdb, to the folder C:\GenMAPP 2 Data\Gene Databases.

GenMAPP Expression Dataset Manager Procedure

• Launch the GenMAPP Program. Check to make sure the correct Gene Database is loaded.
• Select the Data menu from the main Drafting Board window and choose Expression Dataset Manager from the drop-down list. The Expression Dataset Manager window will open.
• Select New Dataset from the Expression Datasets menu. Select the tab-delimited text file that you formatted for GenMAPP (.txt) in the procedure above from the file dialog box that appears.
• The Data Type Specification window will appear. GenMAPP is expecting that you are providing numerical data. If any of your columns has text (character) data, check the box next to the field (column) name.
  • The column StandardName has text data in it, but none of the rest do.
• Allow the Expression Dataset Manager to convert your data.
  • This may take a few minutes depending on the size of the dataset and the computer’s memory and processor speed. When the process is complete, the converted dataset will be active in the Expression Dataset Manager window and the file will be saved in the same folder the raw data file was in, named the same except with a .gex extension; for example, MyExperiment.gex.
  • A message may appear saying that the Expression Dataset Manager could not convert one or more lines of data. Lines that generate an error during the conversion of a raw data file are not added to the Expression Dataset. Instead, an exception file is created. The exception file is given the same name as your raw data file with .EX before the extension (e.g., MyExperiment.EX.txt). The exception file will contain all of your raw data, with the addition of a column named ~Error~. This column contains either error messages or, if the program finds no errors, a single space character.
    • Record the number of errors in your lab notebook.
• Customize the new Expression Dataset by creating new Color Sets which contain the instructions to GenMAPP for displaying data on MAPPs.
  • Color Sets contain the instructions to GenMAPP for displaying data from an Expression Dataset on MAPPs. Create a Color Set by filling in the following different fields in the Color Set area of the Expression Dataset Manager: a name for the Color Set, the gene value, and the criteria that determine how a gene object is colored on the MAPP. Enter a name in the Color Set Name field that is 20 characters or fewer. You will have one Color Set per strain per time point.
  • The Gene Value is the data displayed next to the gene box on a MAPP. Select the column of data to be used as the Gene Value from the drop down list or select [none]. We will use "Avg_LogFC_" for the the appropriate time point.
  • Activate the Criteria Builder by clicking the New button.
  • Enter a name for the criterion in the Label in Legend field.
  • Choose a color for the criterion by left-clicking on the Color box. Choose a color from the Color window that appears and click OK.
  • State the criterion for color-coding a gene in the Criterion field.
    • A criterion is stated with relationships such as "this column greater than this value" or "that column less than or equal to that value". Individual relationships can be combined using as many ANDs and ORs as needed. A typical relationship is

[ColumnName] RelationalOperator Value

with the column name always enclosed in brackets and character values enclosed in single quotes. For example:

[Fold Change] >= 2
[p value] < 0.05
[Quality] = 'high'

This is the equivalent to queries that you performed on the command line when working with the PostgreSQL movie database. GenMAPP is using a graphical user interface (GUI) to help the user format the queries correctly. The easiest and safest way to create criteria is by choosing items from the Columns and Ops (operators) lists shown in the Criteria Builder. The Columns list contains all of the column headings from your Expression Dataset. To choose a column from the list, click on the column heading. It will appear at the location of the cursor in the Criterion box. The Criteria Builder surrounds the column names with brackets.

The Ops (operators) list contains the relational operators that may be used in the criteria: equals ( = ) greater than ( > ), less than ( < ), greater than or equal to ( >= ), less than or equal to ( <= ), is not equal to ( <> ). To choose an operator from the list, click on the symbol. It will appear at the location of the insertion bar (cursor) in the Criterion box. The Criteria Builder automatically surrounds the operators with spaces. The Ops list also contains the conjunctions AND and OR, which may be used to make compound criteria. For example:

[Fold Change] > 1.2 AND [p value] <= 0.05

Parentheses control the order of evaluation. Anything in parentheses is evaluated first. Parentheses may be nested. For example:

[Control Average] = 100 AND ([Exp1 Average] > 100 OR [Exp2 Average] > 100)

Column names may be used anywhere a value can, for example:

[Control Average] < [Experiment Average]

• After completing a new criterion, add the criterion entry (label, criterion, and color) to the Criteria List by clicking the Add button.
  • For the yeast dataset, you will create two criterion for each Color Set. "Increased" will be [<strain>_Avg_LogFC_<timepoint>] > 0.25 AND [<strain>Pval_<timepoint>] < 0.05 and "Decreased will be [<strain>_Avg_LogFC_<timepoint>] < -0.25 AND [<strain>Pval_<timepoint>] < 0.05. Make sure that the increased and decreased average log fold change values match the timepoint of the Color Set.
  • You may continue to add criteria to the Color Set by using the previous steps.
    • The buttons to the right of the list represent actions that can be performed on individual criteria. To modify a criterion label, color, or the criterion itself, first select the criterion in the list by left-clicking on it, and then click the Edit button. This puts the selected criterion into the Criteria Builder to be modified. Click the Save button to save changes to the modified criterion; click the Add button to add it to the list as a separate criterion. To remove a criterion from the list, left-click on the criterion to select it, and then click on the Delete button. The order of Criteria in the list has significance to GenMAPP. When applying an Expression Dataset and Color Set to a MAPP, GenMAPP examines the expression data for a particular gene object and applies the color for the first criterion in the list that is true. Therefore, it is imperative that when criteria overlap the user put the most important or least inclusive criteria in the list first. To change the order of the criteria in the list, left-click on the criterion to select it and then click the Move Up or Move Down buttons. No criteria met and Not found are always the last two positions in the list.
• You will also create two ColorSets to view the ANOVA p values for both strains, with criteria for viewing the unadjusted, Bonferroni-corrected, and B&H corrected p values.
• Save the entire Expression Dataset by selecting Save from the Expression Dataset menu. Changes made to a Color Set are not saved until you do this.
• Exit the Expression Dataset Manager to view the Color Sets on a MAPP. Choose Exit from the Expression Dataset menu or click the close box in the upper right hand corner of the window.
• Upload your .gex file to Lionshare and share it with Dr. Dahlquist. E-mail the link to the file to Dr. Dahlquist.
• Dr. Dahlquist will provide a set of MAPPs with which to view your Expression Dataset. You will select one MAPP that you find interesting to include and interpret for your final lab report.
  • Links to a zipped archive of MAPPs and an Expression Dataset have been e-mailed to your lion e-mail accounts.