Biology Reference
In-Depth Information
3.
Perform spot detection using the “DeCyder spot detection
algorithm 6.0” with a number of 10,000 for “estimated num-
ber of spots.” This number is an overestimation and incorpo-
rates the false positive detection of dust particles and other
disturbing nonprotein-related artifacts.
4. To remove false positives, use the exclusion fi lter “Volume
<30,000.” Other fi lters are usually not needed for good qual-
ity gels.
5.
Visually inspect if spot detection leads to a satisfactory result.
A method to control spot detection quality is to uncheck
“Excluded spots” in the “Spot Display Properties” window
and to ensure that the broad majority of spots are detected and
the majority of very weak spots (typically false positives) are
not detected.
6.
Save the DIA workspace to the corresponding DeCyder project.
The biological variation analysis (BVA) module performs gel-to-gel
spot matching and allows quantitative comparisons between
several groups:
3.6.3. Biological Variation
Analysis
1.
Create a BVA workspace.
2.
Import the DIA workspaces which are to be included in the
BVA analysis.
3.
Add experimental groups in the “Experimental Design View”
of the “Spot Map View.”
4.
Assign the unassigned gel images to the corresponding experi-
mental group.
5.
Select the most representative gel as master gel for matching.
6.
Match the gels with “warping” enabled.
7.
Switch to “Match mode” and check the matching quality man-
ually. If warping has been performed it is helpful to display
warp grids. In addition, color overlay images can be displayed,
where the master image within every gel is presented in yellow
and the standard image is presented in blue.
8.
For gels showing areas with unsatisfactory matching, set land-
marks in the corresponding areas. Alternatively, correct matches
in the corresponding area can be set manually (see Note 13).
9.
Rematch the gels.
When multiple gels have to be analyzed, it is much faster to use the
Batch Processor. After setup, the Batch Processor performs all DIA
and subsequent BVA analyses automatically:
1.
3.6.4. Batch Processing
First, import the cropped gel images into the corresponding
project as described in Subheading 3.6.1 .
2.
Open the Batch Processor module.
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