Agenda

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Agenda

• Intro. of Affy CEL file and CDF file.
• Demo of dChip
• Only performs Li-Wong model.
• Easy to use. Computation is fast.
• Provides good interface and quality assessment.
• Demo of RMAExpress
• Only performs RMA
• Easy to use
• Also provides good interface
• In the demonstration, we will reproduce analysis
  for the 8 arrays (m,n,o,p,q,r,s,t) from Latin
  Square experiment.

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From experiments to analysis (Affymetrix)
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1. CEL files
Type of arrays
Probe intensity information Position (x, y),
mean intensity, standard deviation, of pixels
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1. CEL files

• CEL files record probe intensity information
  for each array.
• The scanner produces a CEL file for each array.
• The CEL file does not contain gene or probe
  annotation information. Such information is
  recorded in CDF file.

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1. CDF files
Array type
Probe set ID
Probe position
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1. CDF files

• CDF files contain probe annotation information
  and mapping of their physical position on the
  array.
• Each array type has a CDF file.
• In bioconductor, the package automatically
  downloads CDF file of the array type that suits
  your CEL files. But in dChip or other program,
  you need to provide the CDF file information.

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Probe analysis of Affymetrix array
CEL files
Array 1
CDF file
Array 2
? ? ?
Array S
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2. dChip

• Need three program or documents
• dChip software (a stand-alone windows program)
• CEL files for each array
• CDF file for the specific array type
• Example
• -- Go to the course web to download the dChip
  software, CEL files of eight arrays from Latin
  Square experiment and the corresponding CDF file.
  Unzip the compressed files to a designated
  directory.

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2. dChip
1. Open program Click on the executable file
dChip2006.exe to open the software.
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2. dChip
2.1. Read in data Read CEL files.
AnalysisgtOpen Group. Put a group name.
Choose the Data directory where your data sets
were saved.
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2. dChip
2.2. Read in data Click on the label (Other
information). Provide location of the CDF file
that suits your array type. Click OK will read
in all CEL files in the directory.
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2. dChip
3. Normalization model AnalysisgtNormalizatio
n Model. An option window will pop up.
Uncheck View normalization plot to ignore the
cumbersome normalization plots and click
Options for more normalization and model
options.
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2. dChip
3. Normalization model You may choose methods
for Backgroudn substraction (choose PM-only
model) and Use selected probes (choose
invariant set). Click OK to start
normalization and model fitting.
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2. dChip
4. Output the expression indexes to a
file ToolsgtExport expression values.
Highlight arrays (on the left) to be exported.
Choose the file name to be saved (the default
saves as .xls but its actually a tab-delimited
text file).
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2. dChip
5. Diagnosis (outlier detection) Click CEL
Image to check the array image. Arrays with
serious quality issue may be marked with black
color on its icon. The problematic probes may
also be marked by flashing white color.
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2. dChip
6. Data matrix (genes on rows and samples on
columns) View the output data matrix (12626 genes
8 samples in our example).
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3. RMA Express preprocessing program

• Go to the website of Ben Bostad group at UC
  Berkeley and download RMA Express program.
  http//rmaexpress.bmbolstad.com/

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3. RMA Express preprocessing program
2. Scroll down to the How do I download and
install it? section and download the newest
version (1.0 release ).

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3. RMA Express preprocessing program
3. Need two files before you run RMA Express, (1)
.CEL file for each array (2) .cdf file for array
type NOTE Go to the course web(http//www.bios
tat.pitt.edu/biost2055/09/index.htm) to download
CEL files of eight arrays from Latin Square
experiment and the corresponding CDF file. Unzip
the compressed files to a designated
directory. (3) You can create a folder to save
only these files. (For example, C//CEL and CDF
file// )

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3. RMA Express preprocessing program
4. How to use RMA Express. If you download it
successfully, you can see the below window first
when you open it.

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3. RMA Express preprocessing program
5. Select to File-gt Read unprocessed files.
Please look at the next slide together. A window
will appear that will ask you to select your .CDF
file. Select it from its location and click
Open Another window will immediately open that
will ask for all .CEL files. Select all .CEL
files in your experiment. (use the shift or
control button to select multiple files).

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3. RMA Express preprocessing program
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3. RMA Express preprocessing program

If you upload those files successfully, you can
see this window in RMA Express.

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3. RMA Express preprocessing program

6. When it is done reading in datafiles, Select
File-gt Compute RMA measure. In the options box
that opens, leave the settings at default
(Background Adjust Yes, Normalization Quantile,
Store residuals unclicked). And then click OK
button.

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3. RMA Express preprocessing program

7. RMA Express will now carry out the analysis.
When finished, it will display Done computing
RMA expression measure. Now select File-gtWrite
Results to file ( log scale or natural scale)
with analysis1.txt file name.

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3. RMA Express preprocessing program

8. In Excel, you can check saved expression file.
( log or natural scale) Please open C//CEL and
CDF files//analysis1.txt