Ebachour Week 8

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General Information about dASH1

We had 15 records total, 4 different time records:

  1. t15: 4 different sets of data
  2. t30: 4 different sets of data
  3. t60: 4 different sets of data
  4. t90: 3 different sets of data

Number of NAs in data: 10,854

Electronic Journal

  • In the Excel spreadsheet, there is a worksheet labeled "Master_Sheet".
    • In this worksheet, each row contains the data for one gene (one spot on the microarray).
    • The first column contains the "MasterIndex", which numbers all of the rows sequentially in the worksheet so that we can always use it to sort the genes into the order they were in when we started.
    • The second column (labeled "ID") contains the gene identifier from the Saccharomyces Genome Database.
    • The third column contains the Standard Name for each of the genes.
    • Each subsequent column contains the log2 ratio of the red/green fluorescence from each microarray hybridized in the experiment (steps 1-5 above having been performed for you already), for each strain starting with wild type and proceeding in alphabetical order by strain deletion.
    • Each of the column headings from the data begin with the experiment name ("wt" for wild type S. cerevisiae data, "dASH1" for the Δash1 data, etc.). "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 "-0", "-1", "-2", etc. after the timepoint.
      • 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).
  • Begin by recording in your wiki, the strain that you will analyze, the filename, the number of replicates for each strain and each time point in your data.
  • NOTE: before beginning any analysis, immediately change the filename so that it contains your initials to distinguish it from other students' work.
  • The first thing you will do is to delete the data columns of the strains that you are not analyzing so that your file contains only the strain's data that you will be working with (to make a smaller and less confusing file).
  • Next you will replace cells that have "NA" in them (which indicates missing data) with an empty cell.
    • Use the keyboard shortcut Control+F to open the "Find" dialog box and select the "Replace" tab.
    • Type "NA" in the Search field and don't type anything in the "Replace" field.
    • Click the button "Replace all" and record the number of replacements made in your electronic lab notebook.
    • Save early and often throughout this protocol. We are working with a large spreadsheet and glitches do occur.

Part 1: Statistical Analysis Part 1

The purpose of the witin-stain ANOVA test is to determine if any genes had a gene expression change that was significantly different than zero at any timepoint.

  1. Create a new worksheet, naming it either "dASH1_ANOVA" as appropriate. For example, you might call yours "wt_ANOVA" or "dHAP4_ANOVA"
  2. Copy the first three columns containing the "MasterIndex", "ID", and "Standard Name" from the "Master_Sheet" worksheet for your strain and paste it into your new worksheet. Copy the columns containing the data for your strain and paste it 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 dASH1_AvgLogFC_(TIME) where dASH1 is your strain designation and (TIME) is 15, 30, etc.
  4. In the cell below the dASH1_AvgLogFC_t15 header, type =AVERAGE(
  5. Then highlight all the data in row 2 associated with dASH1 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 (8) with the t30, t60, t90, and the t120 data.
  9. Now in the first empty column to the right of the dASH1_AvgLogFC_t120 calculation, create the column header dASH1_ss_HO.
  10. In the first cell below this header, type =SUMSQ(
  11. Highlight all the LogFC data in row 2 for your dASH1 (but not the AvgLogFC), press the closing paren key (shift 0),and press the "enter" key.
  12. In the next empty column to the right of dASH1_ss_HO, create the column headers dASH1_ss_(TIME) as in (3).
  13. Make a note of how many data points you have at each time point for your strain. For most of the strains, it will be 4, but for dHAP4 t90 or t120, it will be "3", and for the wild type it will be "4" or "5". Count carefully. Also, make a note of the total number of data points. Again, for most strains, this will be 20, but for example, dHAP4, this number will be 18, and for wt it should be 23 (double-check).
  14. In the first cell below the header dASH1_ss_t15, type =SUMSQ(<range of cells for logFC_t15>)-COUNTA(<range of cells for logFC_t15>)*<AvgLogFC_t15>^2 and hit enter.
    • The COUNTA function counts the number of cells in the specified range that have data in them (i.e., does not count cells with missing values).
    • The phrase <range of cells for logFC_t15> should be replaced by the data range associated with t15.
    • 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 dASH1_ss_t120, create the column header dASH1_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 dASH1_Fstat and dASH1_p-value.
  19. Recall the number of data points from (13): call that total n.
  20. In the first cell of the dASH1_Fstat column, type =((15-4)/4)*(<dASH1_ss_HO>-<dASH1_SS_full>)/<dASH1_SS_full> and hit enter.
    • Don't actually type the n but instead use the number from (13). Also note that "5" is the number of timepoints and the dSWI4 strain has 4 timepoints (it is missing t15).
    • Replace the phrase dASH1_ss_HO with the cell designation.
    • Replace the phrase <dASH1_SS_full> with the cell designation.
    • Copy to the whole column.
  21. In the first cell below the dASH1_p-value header, type =FDIST(<dASH1_Fstat>,5,15-4) replacing the phrase <dASH1_Fstat> with the cell designation and the "n" as in (15) with the number of data points total. (Again, note that the number of timepoints is actually "4" for the dSWI4 strain). 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 dASH1_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 each other 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, dASH1_Bonferroni_p-value.
  2. Type the equation =<dASH1_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 dASH1_Bonferroni_p-value header: =IF(STRAIN_Bonferroni_p-value>1,1,STRAIN_Bonferroni_p-value), where "STRAIN_Bonferroni_p-value" refers to the cell in which the first Bonferroni p value computation was made. 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 "dASH1_ANOVA_B-H".
  2. Copy and paste the "MasterIndex", "ID", and "Standard Name" 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 D.
  4. Select all of columns A, B, C, and D. Sort by ascending values on Column D. Click the sort button from A to Z on the toolbar, in the window that appears, sort by column D, smallest to largest.
  5. Type the header "Rank" in cell E1. 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 E2 and "2" into cell E3. Select both cells E2 and E3. 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 dASH1_B-H_p-value in cell F1. Type the following formula in cell F2: =(D2*6189)/E2 and press enter. Copy that equation to the entire column.
  7. Type "STRAIN_B-H_p-value" into cell G1.
  8. Type the following formula into cell G2: =IF(F2>1,1,F2) and press enter. Copy that equation to the entire column.
  9. Select columns A through G. Now sort them by your MasterIndex in Column A in ascending order.
  10. Copy column G and use Paste special > Paste values to paste it into the next column on the right of your ANOVA sheet.
  • Zip and upload the .xlsx file that you have just created to the wiki.

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 dASH1_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.
    • Unadjusted p value
      • Less than 0.05
        • 1630
        • Percentage= 26.34%
      • Less than 0.01
        • 880
        • Percentage= 14.22%
      • Less than 0.001
        • 356
        • Percentage= 5.75%
      • Less than 0.0001
        • 142
        • Percentage= 2.29%
    • Bonferroni-corrected p value
      • Less than 0.05
        • 53
        • Percentage= 0.856%
    • Benjamin and Hochberg-corrected p value
      • Less than 0.05
        • 730
        • Percentage= 11.80%

I believe that the ACT1 gene did not change expression. I am basing this off the fact that it had smaller average log change values and the larger p values.

Summary Paragraph

When we started our dASH1 analysis, we found that there was the t90 times, directly followed by another set of t90. At first we decided that it was meant to be a t120, so we changed the names and then continued on with our analysis, but then we realized that it was actually just a duplicate of the first t90 set of data, so we ended up just deleted the entire second set of t90.

The data that we were looking at was an experiment to test the effect of cold shock on the expression on all of the genes in the dASH1 strain of yeast. Data was collected at four separate time intervals. The first sixty minutes were under cold shock and the last thirty was the recovery from the cold shock. Our job was to analyze this data by following the script that we were given. I am not really sure what all the data points that we calculated were, but I spoke with Quinn, my partner, to help better understand the data.
He explained to me that the smaller the p value, the more of an indication that the gene displayed change of expression due to the cold shock.

Excel Document and Powerpoint with Values

My spreadsheet
| Our Powerpoint with Values

Acknowledgements

I worked with my homework partner Quinn Lanners in class. We met face-to-face one time outside of class. We texted outside of class and met to work on the excel documents together.
While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.
Ebachour (talk) 17:09, 23 October 2017 (PDT)

References

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Eddie Bachoura

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