Microarray data analysis startup by using GeneSpring

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Microarray data analysis startup by using
GeneSpring

• Bo Gao
• WSU Bioinformatics Core

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Objectives

• Introduce terminology and interface of GeneSpring
• Import data files
• Set up experiments for data analysis
• 1. Normalization
• 2. Parameters
• 3. Interpretations
• 4. Error model
• Perform quality control
• Export gene lists, graphs, clusters, and venn
  diagram

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I. Terminology of GeneSpring
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II. GeneSpring Interface
Genome name
Tool Bar Access to menus and commands of
GeneSpring
Genome Browser Graphically displays the
expression data in the selected gene list
Color Bar Indicate how expression data is being
colored
Navigator Access to data elements relating to
genomes
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1.Tool Bar Accesses GeneSpring Functions
Managing data and images
Provides helpful resources.
Edits Data (i.e. gene lists and experiments)
Managing windows
Display data in the browser.
Improve your genome.
Perform advance analysis i.e. ANOVA, clustering,
and scripts creation.
Setup experiment normalizations,
parameters, interpretations, and samples for
analysis.
Color data with using different criteria.
Perform quality control and data analysis.
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2. Navigator a. Gene List Folder

• A gene list contains collections of genes that
  result from a particular analysis.
• The all genes list is automatically created and
  displayed containing all genes in the genome

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b. Experiments Folder
Experiment Collections of samples that are
analyzed as a set
Interpretation Users instruct genespring how to
treat and display your experiment parameters
Sample Data taken from chips/arrays
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c. Gene List Inspector Double click on a gene
list to see detailed information
The number of genes in the current gene list
Graphically displays the expression data in the
selected gene list
It contains the information about the gene list
creation history
Press the button to display more information
about annotations for the genes
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d. Gene Inspector double click on a gene in a
browser window or a gene in a gene list
Selected gene name
Display information about annotations of the gene
Gene data value
Current gene image
Web links to public databases
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3. Genome Browser
A display of gene list Each line represent a gene
Browser of current setting
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III. Importing Data

• 1. Prepare data for GeneSpring by using a or b,
  and c
• a. Use following steps for getting data from
  compact disk

Create your folder on the desktop.
Open
Copy CAB data file into your folder on the
desktop, then go to c
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b. Use following steps for getting data from
Bioinformatics Core Data Server
Create your folder on the desktop.
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Click on Run
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3
Click on Start
Enter \\bioinfo-serv1.ad.wsu.edu
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1
2
Enter your authentication ID and password
Double click on
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Copy CAB data file into your folder of the desktop
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c. Click on Start ? Programs ? Affymetrix ?
Data Transfer Tool
Select transfer data in to GCOS using CAB files
Click on next
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Set data file location.
Click on CABs
Click on next
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Select a Cab file
Click on
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Click on Yes if want to load the more data.
Click on No if no more data to be loaded in.
Click on
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2. Create Data Report --- Must Do This for Each
File . Open GCOS (GeneChip Operating
Software) . Open a file
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Set Defaults
Click on Grid
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1
Click on Tool
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Click on Defaults
Set Expression
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Click on Autoscale
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Click on OK
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1
Click on Tool
Click on Expression Settings
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Run Analysis
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Click on All Probe Sets
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Enter 125
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Click on OK
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1
Click on Run
Save a file
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Click on Analysis
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Check your data whether or not have a high
background
Use an Excel File Type
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1
Click on Report
2
Choose the file
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If Background Avg. is less than 150 then it is
OK, otherwise, you have to repeat the array.
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Transfer Excel file to a tab delimited file
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Determine a R-Squared Value
Use a Tab Delimited File Type
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Both A2A30 and B2B30 are a data range. You
have to change the data range for your data.
Data come from your replicate chips.
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3. Import data into GeneSpring
Open GeneSpring
Open a genome. Otherwise, import a genome If you
need.
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Open the Tab Delimited File
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Choose a file format
Select a genome Load data into a existing genome
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Add data with using each of your tab delimited
(.txt) file
Select .txt file
Press Add button
Next
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Yes
Create an experiment
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Experiment name
Choose a location
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Normalize Data
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IV. Experiment Normalization
Double click for adding normalization steps
Editing options

• Output of step 1 becomes
• the input of step 2, and so forth
• Click on normalization steps
• and you can move it up or down
• by using editing options

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V. Defining the Experiment Parameters
Editing options
Use the same value for replicate samples
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VI. Creating the Experiment Interpretations
Determine how your experiment should be displayed
Present data in Ratio, Log, or fold-change mode
Display genes on specific flag measurements
Use the error model to associate error with each
measurement
Delete specific conditions from display or
analysis
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VII. Creating the Experiment Error Model
Choose it to specify the parameters that
differentiate the groups of replicates If you
have replicates for each condition of your
experiment
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VIII. Quality Control Elimination of
poor quality samples and genes
Click on Filtering
Click on Advanced Filtering
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Double-click to select all desired filters
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1
Select a gene list
Select a experiment interpretation to filter on
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3
Choose to filter genes on Normalized data, Raw
data, or Control Signal data values
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Set a value of the filter, such as, 100
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1
Select the starting gene list
2
Select conditions 1 and 2 by using the filter
navigator
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Choose a data type
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Select a type of comparison
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Set the folder difference by using the slider or
the text box
Use Add/Remove button to edit condition1 and 2
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Modify the filter logic
Press on the start button
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Experiment name
Choose a location
Gene list
Press the save button
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VIIII. Export Data and Images 1. Export gene
list to MS Excel by using Copy annotated gene list
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Click on Edit
Click on Copy
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Click on Copy Annotated Gene List
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Select annotation fields
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Click on Copy to Clipboard and Paste the list
into MS Excel
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2. Export browser images a. Export graphs
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Click on File
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Click on Save Image
Click on Browser
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Save images as PICT or PNG files
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2
Click on Save
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b. Export clusters
Click on K-means
Click on Tools
Click on Clustering
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2
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Select a gene list
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Select a experiment interpretation
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Click on Save
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Enter your cluster name
Choose a location
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2
3
Click on Save
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c. Create venn diagram . For gene list EXP
gt 100 FC gt 2
Right click on the gene list that you want
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2
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Click on Venn Diagram
Click on Left (Red)
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. For gene list EXP gt 50 FC gt 2.5
Right click on the gene list that you want
1
2
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Click on Venn Diagram
Click on Right (Green)
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. For gene list 99 gt EXP gt 50 FC gt 2
Right click on the gene list that you want
1
2
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Click on Venn Diagram
Click on Bottom (Blue)
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. For all genes
Right click on the gene list that you want
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2
3
Click on Venn Diagram
Click on Universe
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d. Export venn diagram
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Click on File
2
3
Click on Save Image
Click on Venn Diagram
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References

• GeneSpring user manual
• Pam Tang
• Microarray data analysis genespring workshop
• http//www.silicongenetics.com/cgi/SiG.cgi/Support
  /demos/index.smf

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The end