BIOL398-01/S11:Week 11
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This journal entry is due on Tuesday, April 5 at midnight PDT (Wednesday night/Thursday morning). NOTE new due date and that the server records the time as Eastern Daylight Time (EDT). Therefore, midnight will register as 03:00.
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Individual Journal Assignment
- Store this journal entry as "username Week 11" (i.e., this is the text to place between the square brackets when you link to this page).
- Create the following set of links. (HINT: you can do all of this easily by adding them to your template and then using the template on your pages.)
- Link to your journal entry from your user page.
- Link back from your journal entry to your user page.
- Link to this assignment from your journal entry.
- Don't forget to add the "BIOL398-01/S11" category to the end of your wiki page.
Microarray Data Analysis
Background
This is a list of steps required to analyze DNA microarray data.
- Quantitate the fluorescence signal in each spot
- Calculate the ratio of red/green fluorescence
- Log transform the ratios
- Normalize the ratios on each microarray slide
- Steps 1-4 are performed by the GenePix Pro software.
- You will perform the following steps:
- Normalize the ratios for a set of slides in an experiment
- Perform statistical analysis on the ratios
- Compare individual genes with known data
- Steps 5-7 are performed in Microsoft Excel
- Pattern finding algorithms (clustering)
- Map onto biological pathways
- We will use software called STEM for the clustering and mapping
- Create mathematical model of transcriptional network
Each group will analyze a different microarray dataset:
- Wild type data from the Schade et al. (2004) paper you read last week.
- Wild type data from the Dahlquist lab.
- Δgln3 data from the Dahlquist lab.
For your assignment this week, you will keep an electronic laboratory notebook on your individual wiki page that records all the manipulations you perform on the data and the answers to the questions throughout the protocol.
You will download your assigned Excel spreadsheet from LionShare. Because the Dahlquist Lab data is unpublished, please do not post it on this public wiki. Instead, keep the file(s) on LionShare, which is protected by a password.
Experimental Design
On the spreadsheet, 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 match the data from different experiments together later. The second column (labeled "ID") contains the gene identifier from the Saccharomyces Genome Database. Each subsequent column contains the log_{2} ratio of the red/green fluorescence from each microarray hybridized in the experiment (steps 1-4 above having been done for you by the scanner software).
Each of the column headings from the data begin with the experiment name ("Schade" for Schade wild type data, "wt" for Dahlquist wild type data, and "dGLN3" for the Dahlquist Δgln3 data). "LogFC" stands for "Log_{2} Fold Change" which is the Log_{2} 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.
For the Schade data, the timepoints are t0, t10, t30, t120, t12h (12 hours), and t60 (60 hours) of cold shock at 10°C.
For the Dahlquist data (both wild type and Δgln3), 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). Note that the experimental designs are different.
- Begin by recording in your wiki the number of replicates for each time point in your data. For the group assigned to the Schade data, compare the number of replicates with what is stated in the Materials and Methods for the paper. Is it the same? If not, how is it different?
Between-chip Normalization
To scale and center the data (between chip normalization) perform the following operations:
- Insert a new Worksheet into your Excel file, and name it "scaled_centered".
- Go back to the "compiled_raw_data" worksheet, Select All and Copy. Go to your new "scaled_centered" worksheet, click on the upper, left-hand cell (cell A1) and Paste.
- Insert two rows in between the top row of headers and the first data row.
- In cell A2, type "Average" and in cell A3, type "StdDev".
- You will now compute the Average log ratio for each chip (each column of data). In cell C2, type the following equation:
=AVERAGE(C4:C6190)
and press "Enter". Excel is computing the average value of the cells specified in the range given inside the parentheses. Instead of typing the cell designations, you can left-click on the beginning cell (let go of the mouse button), scroll down to the bottom of the worksheet, and shift-left-click on the ending cell.
- You will now compute the Standard Deviation of the log ratios on each chip (each column of data). In cell B3, type the following equation:
=STDEV(C4:C6190)
and press "Enter".
- Excel will now do some work for you. Copy these two equations (cells C2 and C3) and paste them into the empty cells in the rest of the columns. Excel will automatically change the equation to match the cell designations for those columns.
- You have now computed the average and standard deviation of the log ratios for each chip. Now we will actually do the scaling and centering based on these values.
- Insert a new column to the right of each data column and label the top of the column as with the same name as the column to the left, but adding "_sc" for scaled and centered to the name. For example, "wt_LogFC_t15-1_sc"
- In cell D4, type the following equation:
=(C4-$C$2)/$C$3
In this case, we want the data in cell C4 to have the average subtracted from it (cell C2) and be divided by the standard deviation (cell C3). We use the dollar sign symbols surrounding the "C" to tell Excel to always reference that cell in the equation, even though we will paste it for the entire column. Why is this important?
- Copy and paste this equation into the entire column.
- Repeat the scaling and centering equation for each of the columns of data. Be sure that your equation is correct for the column you are calculating.
Shared Journal Assignment
- Store your journal entry in the shared Class Journal Week 11 page. If this page does not exist yet, go ahead and create it (congratulations on getting in first :) )
- Link to your journal entry from your user page.
- Link back from the journal entry to your user page.
- Sign your portion of the journal with the standard wiki signature shortcut (
~~~~
). - Add the "BIOL398-01/S11" category to the end of the wiki page (if someone has not already done so).