Moneil5 Week 11

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Margaret J. ONeil

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Purpose

The purpose of this assignment is to familiarize ourselves with how microarray data from our papers was analyzed using raw data collected from the Dahlquist lab. The purpose of this week's assignment is to also learn about the important of reproducibility in data, as we are working with data sets following clearly outlined protocols of what was done before.

Workflow/Methods/Results

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
    • Steps 1-3 have been performed for you by the GenePix Pro software (which runs the microarray scanner).
  4. Normalize the ratios on each microarray slide
  5. Normalize the ratios for a set of slides in an experiment
  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)
  9. Map onto biological pathways
    • We will use software called STEM for the clustering and mapping
  10. Identifying regulatory transcription factors responsible for observed changes in gene expression
  11. Dynamical systems modeling of the gene regulatory network
    • The modeling will be performed in MATLAB

Statistical Analysis Part 1: ANOVA

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 "wt_ANOVA" as appropriate.
  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 wt_AvgLogFC_(TIME) where (TIME) is 15, 30, etc.
  4. In the cell below the wt_AvgLogFC_t15 header, type =AVERAGE(
  5. Then highlight all the data in row 2 associated with wt 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 wt_AvgLogFC_t120 calculation, create the column header wt_ss_HO.
  10. In the first cell below this header, type =SUMSQ(
  11. Highlight all the LogFC data in row 2 for wt (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 wt_ss_HO, create the column headers wt_ss_(TIME) as in (3).
  13. Make a note of how many data points you have at each time point for your strain. For the wild type it will be "4" or "5". Count carefully. Also, make a note of the total number of data points. For wt it should be 23 (double-check).
  14. In the first cell below the header wt_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 wt_ss_t120, create the column header wt_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 wt_Fstat and wt_p-value.
  19. Recall the number of data points from (13): call that total n.
  20. In the first cell of the wt_Fstat column, type =((n-5)/5)*(<wt_ss_HO>-<wt_SS_full>)/<wt_SS_full> and hit enter.
    • Don't actually type the n but instead use the number from (13).
    • Replace the phrase wt_ss_HO with the cell designation.
    • Replace the phrase <wt_SS_full> with the cell designation.
    • Copy to the whole column.
  21. In the first cell below the wt_p-value header, type =FDIST(<wt_Fstat>,5,n-5) replacing the phrase <wt_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 wt_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, wt_Bonferroni_p-value.
  2. Type the equation =<wt_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 wt_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 "wt_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 wt_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 "wt_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.
  • Upload the .xlsx file that you have just created to Box. Send Dr. Dahlquist and Dr. Fitzpatrick an e-mail with the link to the file (e-mail kdahlquist or bfitzpatrick 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 wt_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)?
      2600 genes have p <0.05, which is 42% of the total genes
    • How many genes have p < 0.01? and what is the percentage (out of 6189)?
      1727 genes have p < 0.01, which is 27.9% of the total genes
    • How many genes have p < 0.001? and what is the percentage (out of 6189)?
      1015 genes have p < 0.01, which is 16.4% of the total genes
    • How many genes have p < 0.0001? and what is the percentage (out of 6189)?
      574 genes have p < 0.0001, which is 9.27% of the total genes
  • 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)?
      302 genes have a Bonferroni-corrected p-value of p < 0.05. This is 4.88% of the total genes.
    • How many genes are p < 0.05 for the Benjamini and Hochberg-corrected p value? and what is the percentage (out of 6189)?
      1936 genes have a Benjamini and Hochberg-corrected p value of p < 0.05, which is 31.28% of the total genes
  • 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 "wt_AvgLogFC_(TIME)" in step 3 of the ANOVA analysis.
    The unadjusted p-value is 5.18E-12, Bonferonni-corrected is 3.21E-08, and B-H-corrected is 1.88E-09. The average Log fold change at t=15 is 3.2792, at t=30 is 3.621, at t=60 is 3.5265, at t=90 is -2.0498, and at t=120 is -0.6062.
  • We will compare the numbers we get between the wild type strain and the other strains studied, organized as a table. Use this sample PowerPoint slide to see how your table should be formatted.

Clustering and GO Term Enrichment 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 "Master_Index". Rename this column to "SPOT". Column B should be named "ID". Rename this column to "Gene Symbol". Delete the column named "Standard_Name".
      • 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.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 OpenWetWare and link to them on your individual journal page. (Note that 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 OpenWetWare and link to them on your individual journal page. (Note that 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 cold shock timepoints. Each member of your group should choose a different profile. Answer the following:
      • Why did you select this profile? In other words, why was it interesting to you?
        I selected profile 0 because the graph curve was interesting. It is the only significant profile I have where the expression change is overall negative and gets more negative over time.
      • How many genes belong to this profile?
        53 genes belong to this profile
      • How many genes were expected to belong to this profile?
        32.7 genes were assigned
      • 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.
      • The p-value is 5.80E-04, which is less than 0.05, which makes the profile significant.
      • 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? - 17 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? - 2 GO terms are associated with this profile at corrected p < 0.05.
      • Select 6 Gene Ontology terms from your filtered list (either p < 0.05 or corrected p < 0.05).
        • Each member of the group will be reporting on his or her own cluster in your presentation next week. You should take care to choose terms that are the most significant, but that are also not too redundant. For example, "RNA metabolism" and "RNA biosynthesis" are redundant with each other because they mean almost the same thing.
          • GO:0072599 establishment of protein localization to endoplasmic reticulum
          • GO:0044092 negative regulation of molecular function
          • GO:0006457 protein folding
          • GO:0044427 chromosomal part
          • GO:0033554 cellular response to stress
          • GO:0016817 hydrolase activity, acting on acid anhydrides
          • Note whether the same GO terms are showing up in multiple clusters.
        • Look up the definitions for each of the terms at http://geneontology.org. In your final presentation, 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? Also, what does this have to do with the transcription factor being deleted (for the Δgln3 and Δswi4 groups)?
        • 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 center top 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.
          • GO:0072599 establishment of protein localization to endoplasmic reticulum
            Defn. The directed movement of a protein to a specific location in the endoplasmic reticulum. Source: GOC:mah
          • GO:0044092 negative regulation of molecular function
            Defn. Any process that stops or reduces the rate or extent of a molecular function, an elemental biological activity occurring at the molecular level, such as catalysis or binding. Source: GO:jl
          • GO:0006457 protein folding
            Defn. The process of assisting in the covalent and noncovalent assembly of single chain polypeptides or multisubunit complexes into the correct tertiary structure. Source: GOC:go_curators, GOC:rb
          • GO:0044427 chromosomal part
            Defn. Any constituent part of a chromosome, a structure composed of a very long molecule of DNA and associated proteins (e.g. histones) that carries hereditary information. Source: GOC:jl
          • GO:0033554 cellular response to stress
            Defn. Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating the organism is under stress. The stress is usually, but not necessarily, exogenous (e.g. temperature, humidity, ionizing radiation). Source: GOC:mah
          • GO:0016817 hydrolase activity, acting on acid anhydrides
            Defn. Catalysis of the hydrolysis of any acid anhydride. Source: GOC:jl

Results/Data Files

Data Files

Question Answers from Protocol

From "Sanity Check"

  • How many genes have p < 0.05? and what is the percentage (out of 6189)?
    2600 genes have p <0.05, which is 42% of the total genes
  • How many genes have p < 0.01? and what is the percentage (out of 6189)?
    1727 genes have p < 0.01, which is 27.9% of the total genes
  • How many genes have p < 0.001? and what is the percentage (out of 6189)?
    1015 genes have p < 0.01, which is 16.4% of the total genes
  • How many genes have p < 0.0001? and what is the percentage (out of 6189)?
    574 genes have p < 0.0001, which is 9.27% of the total genes
  • How many genes are p < 0.05 for the Bonferroni-corrected p value? and what is the percentage (out of 6189)?
    302 genes have a Bonferroni-corrected p-value of p < 0.05. This is 4.88% of the total genes.
  • How many genes are p < 0.05 for the Benjamini and Hochberg-corrected p value? and what is the percentage (out of 6189)?
    1936 genes have a Benjamini and Hochberg-corrected p value of p < 0.05, which is 31.28% of the total genes
  • 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 "wt_AvgLogFC_(TIME)" in step 3 of the ANOVA analysis.
    The unadjusted p-value is 5.18E-12, Bonferonni-corrected is 3.21E-08, and B-H-corrected is 1.88E-09. The average Log fold change at t=15 is 3.2792, at t=30 is 3.621, at t=60 is 3.5265, at t=90 is -2.0498, and at t=120 is -0.6062.

From "Analyzing and Interpreting STEM Results"

  • Why did you select this profile? In other words, why was it interesting to you?
    I selected profile 0 because the graph curve was interesting. It is the only significant profile I have where the expression change is overall negative and gets more negative over time.
  • How many genes belong to this profile?
    53 genes belong to this profile
  • How many genes were expected to belong to this profile?
    32.7 genes were assigned
  • What is the p value for the enrichment of genes in this profile?
    The p-value is 5.80E-04, which is less than 0.05, which makes the profile significant.
  • How many GO terms are associated with this profile at p < 0.05?
    17 GO terms are associated with this profile at p < 0.05.
  • How many GO terms are associated with this profile with a corrected p value < 0.05?
    2 GO terms are associated with this profile at corrected p < 0.05.
  • Look up the definitions for each of the terms.
    • GO:0072599 establishment of protein localization to endoplasmic reticulum
      Defn. The directed movement of a protein to a specific location in the endoplasmic reticulum. Source: GOC:mah
    • GO:0044092 negative regulation of molecular function
      Defn. Any process that stops or reduces the rate or extent of a molecular function, an elemental biological activity occurring at the molecular level, such as catalysis or binding. Source: GO:jl
    • GO:0006457 protein folding
      Defn. The process of assisting in the covalent and noncovalent assembly of single chain polypeptides or multisubunit complexes into the correct tertiary structure. Source: GOC:go_curators, GOC:rb
    • GO:0044427 chromosomal part
      Defn. Any constituent part of a chromosome, a structure composed of a very long molecule of DNA and associated proteins (e.g. histones) that carries hereditary information. Source: GOC:jl
    • GO:0033554 cellular response to stress
      Defn. Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating the organism is under stress. The stress is usually, but not necessarily, exogenous (e.g. temperature, humidity, ionizing radiation). Source: GOC:mah
    • GO:0016817 hydrolase activity, acting on acid anhydrides
      Defn. Catalysis of the hydrolysis of any acid anhydride. Source: GOC:jl

Conclusions

From this data, it can be seen that some genes play a larger role in cold shock response than others, in terms of the significant difference in their activation at the time of cold shock and cold shock recovery. It can also be seen that different clusters of genes might be playing a role in this cold shock response, which would be interesting to compare across clusters, and eventually across deletion strains to determine which genes might be playing the largest role in regulating the response to cold shock.

References

Dahlquist, Kam D. (2017) BIOL398-05/S17:Week 11. Retrieved from http://www.openwetware.org/wiki/BIOL398-05/S17:Week_11 on 4 April 2017.

Acknowledgments

  • Worked on STEM with Lauren Kelly on Tuesday, March 4, 2017.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Margaret J. Oneil 00:44, 5 April 2017 (EDT)

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