Dramir36 Week 8

Quantitate the fluorescence signal in each spot
Calculate the ratio of red/green fluorescence
Log₂ transform the ratios
- Steps 1-3 have been performed for you by the GenePix Pro software (which runs the microarray scanner).
Normalize the ratios on each microarray slide
Normalize the ratios for a set of slides in an experiment
- Steps 4-5 was performed for you using a script in R, a statistics package (see: Microarray Data Analysis Workflow)
- You will perform the following steps:
Perform statistical analysis on the ratios
Compare individual genes with known data
- Steps 6-7 are performed in Microsoft Excel
Pattern finding algorithms (clustering)
Map onto biological pathways
- We will use software called STEM for the clustering and mapping
Identifying regulatory transcription factors responsible for observed changes in gene expression
Dynamical systems modeling of the gene regulatory network (GRNmap)
Viewing modeling results in GRNsight

Notes/Methods/Results

The strain that will be analyzed is dHAP4. There are four replicates for the 15,30, and 60 minute time points, but only three replicates for the 90 and 120 minute time points.

T test: is this gene expression change significantly different than zero at a time point?

p>0.05 5%

probability that you would have seen at least this big of a change by chance.

ANOVA: is the gene expression significantly different than zero at any time point?

Values below 0.25 should be considered to be a gene with no change in expression

Experimental Design and Getting Ready

The data used in this exercise is publicly available at the NCBI GEO database in record GSE83656.

Begin by downloading the Excel file for dHAP4, found in the "Data/Files" section of this page
In the Excel spreadsheet, there is a worksheet labeled "Master_Sheet_dHAP4"
- 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 Systematic Name (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, dHAP4. "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).

Statistical Analysis Part 1: ANOVA

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

Create a new worksheet, naming it "dHAP4_ANOVA".
Copy the first three columns containing the "MasterIndex", "ID", and "Standard Name" from the "Master_Sheet" worksheet for dHAP4 and paste it into your new worksheet. Copy the columns containing the data for dHAP4 and paste it into your new worksheet.
At the top of the first column to the right of your data, create five column headers of the form dHAP4_AvgLogFC_(TIME) where (TIME) is 15, 30, etc.
In the cell below the dHAP4_AvgLogFC_t15 header, type =AVERAGE(
Then highlight all the data in row 2 associated with t15, press the closing paren key (shift 0),and press the "enter" key.
This cell now contains the average of the log fold change data from the first gene at t=15 minutes.
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.
Repeat steps (4) through (8) with the t30, t60, t90, and the t120 data.
Now in the first empty column to the right of the dHAP4_AvgLogFC_t120 calculation, create the column header dHAP4_ss_HO.
In the first cell below this header, type =SUMSQ(
Highlight all the LogFC data in row 2 (but not the AvgLogFC), press the closing paren key (shift 0),and press the "enter" key.
In the next empty column to the right of dHAP4_ss_HO, create the column headers dHAP4_ss_(TIME) as in (3).
Make a note of how many data points you have at each time point for dHAP4. 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. dHAP4 will be 18 (double-check).
In the first cell below the header dHAP4_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.
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.
In the first column to the right of dHAP4_ss_t120, create the column header dHAP4_SS_full.
In the first row below this header, type =sum(<range of cells containing "ss" for each timepoint>) and hit enter.
In the next two columns to the right, create the headers dHAP4_Fstat and dHAP4_p-value.
Recall the number of data points from (13): call that total n.
In the first cell of the dHAP4_Fstat column, type =((n-5)/5)*(<dHAP4_ss_HO>-<dHAP4_SS_full>)/<dHAP4_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.
- Replace the phrase dHAP4_ss_HO with the cell designation.
- Replace the phrase <dHAP4_SS_full> with the cell designation.
- Copy to the whole column.
In the first cell below the dHAP4_p-value header, type =FDIST(<dHAP4_Fstat>,5,n-5) replacing the phrase <dHAP4_Fstat> with the cell designation and the "n" as in (13) with the number of data points total. Copy to the whole column.
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 dHAP4_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

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, dHAP4_Bonferroni_p-value.
Type the equation =<dHAP4_p-value>*6189, Upon completion of this single computation, use the Step (10) trick to copy the formula throughout the column.
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 dHAP4_Bonferroni_p-value header: =IF(dHAP4_Bonferroni_p-value>1,1,dHAP4_Bonferroni_p-value), where "dHAP4_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

Insert a new worksheet named "dHAP4_ANOVA_B-H".
Copy and paste the "MasterIndex", "ID", and "Standard Name" columns from your previous worksheet into the first two columns of the new worksheet.
For the following, use Paste special > Paste values. Copy your unadjusted p values from your ANOVA worksheet and paste it into Column D.
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.
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.
Now you can calculate the Benjamini and Hochberg p value correction. Type dHAP4_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.
Type "dHAP4_B-H_p-value" into cell G1.
Type the following formula into cell G2: =IF(F2>1,1,F2) and press enter. Copy that equation to the entire column.
Select columns A through G. Now sort them by your MasterIndex in Column A in ascending order.
Copy column G and use Paste special > Paste values to paste it into the next column on the right of your ANOVA sheet.

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 dHAP4_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)?

There are 2,479 genes that have p < 0.05, which is 40% of the total gene data set.

- How many genes have p < 0.01? and what is the percentage (out of 6189)?

There are 1,583 genes that have p < 0.01, which is 25.57% of the total gene data set.

- How many genes have p < 0.001? and what is the percentage (out of 6189)?

There are 739 genes that have p < 0.001, which is 11.94% of the total gene data set.

- How many genes have p < 0.0001? and what is the percentage (out of 6189)?

There are 280 genes that have p < 0.0001, which is 4.5% of the total gene data set.

- Note that it is a good idea to create a new worksheet in your workbook to record the answers to these questions. Then you can write a formula in Excel to automatically calculate the percentage for you.
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)?

There are 75 genes that have p < 0.05, which is 1.21% of the total gene data set.

- How many genes are p < 0.05 for the Benjamini and Hochberg-corrected p value? and what is the percentage (out of 6189)?

There are 1735 genes that have p < 0.05, which is 28% of the total gene data set.

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.
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. Upload your slide to the wiki.
- Note that since the wild type data is being analyzed by one of the groups in the class, it will be sufficient for this week to supply just the data for your strain. We will do the comparison with wild type at a later date.
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?

The unadjusted p-value is 0.016364209, the Bonferroni-corrected p-value is 101.2780918, and the B-H-corrected p-value is 0.05552527.

What is its average Log fold change at each of the timepoints in the experiment?

The average Log fold change at 15 min is 2.69945, 3.2508 at 30 min, 3.519975 at 60 min, -1.100566667 at 90 min, and -1.797666667 at 120 min.

Note that the average Log fold change is what we called "STRAIN)_AvgLogFC_(TIME)" in step 3 of the ANOVA analysis. Does NSR1 change expression due to cold shock in this experiment?

NSR1 changes expression after the 90 min time interval.

For fun, find "your favorite gene" (from your Week 3 assignment) in the 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?

The CDC28 gene has an unadjusted p-value of 0.07016077, a Bonferroni-corrected p-value of 434.2250052, and a B-H-corrected p-value of 0.157842605.

Does your favorite gene change expression due to cold shock in this experiment?

CDC28 did not change expression because the p-value was not less than 0.05.

Clustering and GO Term Enrichment with stem (part 2)

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.
Now download and extract the STEM software. Click here to go to the STEM web site.
- Click on the download link 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.
  - You now need to download the Gene Ontology and yeast GO annotations and place them in this folder.
  - Click here to download the file "gene_ontology.obo".
  - Click here to download the file "gene_association.sgd.gz".
- Inside the folder, double-click on the stem.jar to launch the STEM program.
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, leave the default selection for the three drop-down menu selections for Gene Annotation Source, Cross Reference Source, and Gene Location Source as "User provided".
3. Click the "Browse..." button to the right of the "Gene Annotation File" item. Browse to your "stem" folder and select the file "gene_association.sgd.gz" and click Open.
4. 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.
5. In section 4 (Execute) click on the yellow Execute button to run STEM.
  - This is the stopping point for the Week 8 assignment.
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 the wiki 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 the wiki 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).
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?
  - 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 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 research presentation. 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.
      - 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 research 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 groups working with deletion strain data)?
    - 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 on the left of the page.
    - 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.

Data/Files

Conclusion

Acknowledgments

Copied purpose and methods/procedure from Week 7 and Week 8 assignment page to individual journal and modified steps to relate to the dHAP4 data

References

Dramir36 Week 8

Contents

Purpose

Background

Notes/Methods/Results

Experimental Design and Getting Ready

Statistical Analysis Part 1: ANOVA

Calculate the Bonferroni and p value Correction

Calculate the Benjamini & Hochberg p value Correction

Sanity Check: Number of genes significantly changed

Clustering and GO Term Enrichment with stem (part 2)

Data/Files

Conclusion

Acknowledgments

References

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