DeCyder Differential Analysis Software v5.0

DeCyder Differential Analysis Software v5.0
data file
DeCyder Differential Analysis Software v5.0
Differential Analysis Software designed for use in 2-D Fluorescence Difference Gel Electrophoresis
DeCyder™ Differential Analysis Software has been
specifically developed for 2-D Fluorescence Difference Gel
Electrophoresis (2-D DIGE). DeCyder software is the result
of a pioneering and unique approach to 2-D image analysis.
For the first time in the history of 2-D electrophoresis, an
internal standard can be included for every spot on every
2-D gel to give the most accurate quantitation possible.
DeCyder software offers:
• Automatic analysis: significantly reduces hands-on time
• No within-gel spot matching is necessary: eliminates errors
• Automatic presentation of spot statistics
• Internal standard approach: increases accuracy and simplifies
gel-to-gel matching
Traditional 2-D gel electrophoresis is a well-established
technique for protein analysis, though time-consuming and
labor-intensive. Many gels have to be run, analyzed, and
compared. However, the reproducibility between gels and
significant system variability make it difficult to distinguish
between system and inherent biological variation, which
means that differences in protein abundance can rarely be
confidently predicted.
DeCyder software consists of a fully automated image
analysis software suite that enables the detection,
quantitation, matching, and analysis of 2-D DIGE gels. The
software is compatible with CyDye™ DIGE Fluor minimal
dyes and saturation dyes from the scarce sample labelling
kit. Using DeCyder software, expression differences
identified by 2-D DIGE can be confidently assigned to
induced biological change and are not due to system
variability. Every difference is assigned a statistical confidence
DeCyder is unique in fully exploiting the advantages offered
by multiplexing, the co-migration of more than one sample
per gel, by allowing the inclusion of an internal standard.
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Fig 1. Overview of 2-D DIGE workflow (three-dye example).
Multiplexing is performed using proprietary size- and
charge-matched CyDye DIGE Fluor minimal or saturation
dyes to label protein samples. Up to three samples can be
separated on the same 2-D gel, which is then scanned with
the Typhoon™ 9400 series Variable Mode Imager. Using
DeCyder software, very small differences in protein
expression can be measured with a high degree of confidence.
Use of an internal standard effectively eliminates gel-to-gel
variation. Detection of differences in expression of less than
10%, with over 95% confidence, can be achieved within
An overview of the 2-D DIGE workflow is shown in Figure 1.
DeCyder Differential Analysis Software v5.0 suite consists of
several modules:
Batch Processor: For automated detection, quantitation
matching, and comparison of multiple 2-D DIGE gels.
Differential In-gel Analysis (DIA): For co-detection,
background subtraction, normalization, and quantitation of
spots in an image pair.
Biological Variation Analysis (BVA): For matching multiple
gels for comparison and statistical analysis of proteinabundance changes.
XML Toolbox: For exporting spot data from DIA or BVA
modules for further downstream analysis.
Batch Processor
Fig 2. Each screen quadrant is linked in the DIA user interface.
The Batch Processor allows unattended and automated
detection, quantitation, matching and comparison of
multiple 2-D DIGE gels. The Batch Processor links both the
DeCyder DIA and BVA modules to perform spot detection
and inter-gel matching of multiple gel images. Several
hundred image pairs per experiment, with up to three dyes
per gel, can be automatically processed without user
Gel images are first processed in the DeCyder DIA module to
identify and quantitate protein spots. The DIA module
generates a series of XML files containing information on all
the protein spots on each of the gels. These files are then
used for inter-gel matching and subsequent analyses in the
other DeCyder software modules.
Differential In-gel Analysis (DIA)
DIA detects up to three images in a gel creating a single
overlay. Co-detection of an image pair (internal standard
and one sample) utilizes novel algorithms to provide a
consistent and accurate ratio measurement. Background
subtraction, quantitation, normalization, and first-level-ofmatching (within gel) are automated for high-throughput
analysis with low experimental variation.
The user interface is divided into quadrants: Gel view,
Histogram view, 3-D view, and Spot Table view. Each of these
views is linked in such a way that selecting a spot in one view
will display information on the same spot in the other views
(Fig 2).
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The DIA module processes a pair of images from a single gel.
The internal standard is loaded as the primary image
followed by the secondary image, derived for example from
either a control or treated sample. Spot analysis is carried
out on the loaded images.
The detection algorithm is designed to take advantage of the
co-migration benefits of Ettan™ DIGE CyDye fluors. The
pair of co-run images is merged together, incorporating all
spot features in a single image. Spot detection and spot
boundary definition are performed using pixel data from the
individual raw images and the merged image. The resultant
spot map is overlaid onto the original two image files. Since
the spot boundaries are identical for both images, they are
effectively already matched, streamlining the process and
resulting in more accurate volume calculations.
The DIA module measures spot protein abundance for the
secondary image. The quantitation is expressed as a spot
ratio, comparing spot volumes on the secondary image with
corresponding spot volumes of the internal standard.
Linking every sample to a common internal standard makes
direct comparison of protein expression levels between
multiple gels easier and more accurate.
Biological Variation Analysis (BVA)
Images processed through the Batch Processor are matched
between gels using the BVA feature, which detects the
consistency of differences between samples across all the gels
and applies statistics to associate a level of confidence for
each of the differences.
The BVA user interface is divided into four linked
quadrants, as in the DIA user interface (Fig3a). In addition
to these four views, there are also four screens displaying
different aspects of the gel-to-gel matching data. A further
screen view can be selected in the top right quadrant and
allows the user to view the experimental design. (Fig 3b).
Fig 3b. BVA interface showing the EDSA screen view.
BVA has four principle group comparison methods that can
be applied to analyze protein spot data:
• Average ratio between two groups or two populations of groups
• Student’s t-test analysis between two groups or two populations
of groups
• One-way ANOVA (ANalysis Of VAriance) for statistical analysis
between all groups
• Two-way ANOVA for statistical analysis between groups or
populations of groups in an experimental design where there
are two independent factors such as in a time-dose study. This
analysis allows the internal and mutual effects of the two factors
to be quantitated.
Fig 3a. BVA user interface is used to detect and statistically
quantitate differences between samples.
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The log standardized abundance is the only variable
subjected to the above statistical analyses within DeCyder
BVA. It is derived from the normalized spot volume,
standardized against the intra-gel standard. Log values are
used so that the data points approach a normal distribution
around zero to fulfil the requirements of subsequent
statistical tests. Consequently, the statistical analysis
functionality is not valid unless the experimental design
includes an internal standard on every gel.
Using XML files generated by the DIA analyses, the BVA
module carries out inter-gel and statistical analyses. Spot
maps are assigned to experimental groups to allow statistical
analyses to be performed. All spot maps are matched to a
master image that is either automatically assigned to the spot
map with the highest number of detected spots, or user
defined. Spot-matching uses a pattern-recognition algorithm
that automatically matches one single spot in one gel to a
single spot in another gel, based on its neighboring spots.
DeCyder software has an EDSA-setting feature for fully
automated statistical analysis. A list of statistically significant
differences is automatically generated, together with an
optional spot-picking list for further analysis. The spot
picking boundary is automatically defined and can be viewed
in the BVA screen. Where two spots are close together, the
software allows the user to edit the position of the picking
boundary to increase the accuracy of picking the protein of
The BVA module is used to establish the significance of
changes in expression of specific proteins from different
experimental groups. The software performs the appropriate
statistical calculations and displays the results in the Protein
table. The graph view shows the differences between the
different experimental groups, providing information about
the spread, and therefore the consistency of data between the
The internal standard is created by pooling aliquots of all
biological samples in the experiment. This standard is
labelled with one CyDye DIGE Fluor dye and run together
with individual samples (Table 1). Samples are thereby in-gel
linked to a common standard, giving accurate quantitation
and separating gel-to-gel from inherent biological variation.
The statistical analyses can be used to select proteins for
picking, digestion, and subsequent analysis by mass
spectrometry. Selection of protein spots can be based on
multiple criteria such as statistical significance of change,
magnitude of change, and spot volume.
XML Toolbox
Data generated in both the DIA and BVA modules can be
saved as XML files. These files contain all information on the
processing of the images throughout the entire procedure.
XML Toolbox comprises different tools for the extraction of
data from the different XML files produced by DeCyder
software to enable custom-designed conversion of data into
text files, HTML files, or other data formats. Two basic tools
are supplied to create Tabbed text files and Web tables. The
XML format can be used to transfer data between the
different modules of DeCyder software, and to make data
available for post-processing.
Importance of internal standardization
The use of appropriate experimental design and statistical
analysis (EDSA) is central to 2-D DIGE technology and
ensures the best possible results. The recommended EDSA
uses an internal pooled standard approach, removing the
system variable associated with gel-to-gel variation. This
methodology is unique to 2-D DIGE. An example of such an
experimental design is shown in Table 1.
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Table 1. An example of experimental design implemented in
DeCyder Differential Analysis Software to derive statistical data
on differences between control and treated samples labelled
with both CyDye DIGE Fluor minimal and saturation dyes.
Gel number
Pooled standard
Control 1
Treated 1
Pooled standard
Control 2
Treated 2
Pooled standard
Control 3
Treated 3
Pooled standard
Treated 4
Control 4
Pooled standard
Treated 5
Control 5
Pooled standard
Treated 6
Control 6
Example of experimental design for CyDye DIGE Fluor minimal
Gel number
Pooled standard
Treated 1
Pooled standard
Treated 2
Pooled standard
Treated 3
Pooled standard
Treated 4
Pooled standard
Treated 5
Pooled standard
Treated 6
Pooled standard
Control 1
Pooled standard
Control 2
Pooled standard
Control 3
Pooled standard
Control 4
Pooled standard
Control 5
Pooled standard
Control 6
Example of experimental design using CyDye DIGE Fluor
saturation dyes from the CyDye DIGE Fluor Labelling Kit for
Scarce Samples
2-D DIGE has very low experimental variation therefore
biological replicates can be used without needing replicates
of the same sample. Using conventional 2-D electrophoresis
the experiment in Table 1 would require a minimum of three
replicates of each control and treated sample to calculate the
system variation making a total of 36 gels compared to the
six required with 2-D DIGE.
PC requirements
Operating System:
Video card:
Color resolution:
Screen resolution:
Virtual memory
Windows™ XP Professional
Pentium™ 4 processor, 1.5 GHz
Minimum 1 GB
Capable of 32 bit color.
Video card driver needs to support
Open GL (v1.2 or later) – ensure the latest
compatible driver is installed.
Set to 32 bit color.
Set to 1024 x 768 pixels, landscape
(with 24 bits Z-buffer/preferably 32 bits).
Set so that the total amount of available
memory, including physical RAM, is greater
than 2.5 GB.
Internet Explorer™ v5.5 or higher must be
installed to run the XML Toolbox module.
Note: Avoid running other programs at the
same time as the various DeCyder
Differential Analysis Software modules.
File specifications
DeCyder software is compatible with 16-bit TIFF images and
*.GEL format files up to a maximum image size of 20 MB.
File output is in XML format to allow easy parsing of data
for archiving or downstream analysis. DeCyder software is
optimized for use with Typhoon 9000 series Variable Mode
Imager for file management of multiplexed gels.
18-1164-85 AB, 2003-06 • p5
Ordering Information
Code no.
DeCyder Differential Analysis Software
v5.0 including PC and single user license
(minimum computer specification
1.5 GHz processor, 1024 MB memory) 1
DeCyder Differential Analysis Software
v5.0 including single user license
Additional DeCyder Differential
Analysis Software v5.0 user license
DeCyder Differential Analysis Software
upgrade kit from v4.0 to v5.0
Related products
Code no.
CyDye DIGE Fluor Cy2 minimal dye, 10 nmol
CyDye DIGE Fluor Cy2 minimal dye, 25 nmol
CyDye DIGE Fluor Cy3 minimal dye, 10 nmol
CyDye DIGE Fluor Cy3 minimal dye, 25 nmol
CyDye DIGE Fluor Cy5 minimal dye, 10 nmol
CyDye DIGE Fluor Cy5 minimal dye, 25 nmol
CyDye DIGE Fluor Labelling Kit for Scarce Samples
(For a mimimum of 12 labelling reactions)
CyDye DIGE Fluor Labelling Kit for Scarce Samples
and Preparative Gel Labelling (For a minimum of
12 labelling reactions and 1 prep gel)
Typhoon 9400 Variable Mode Imager with PC
Typhoon 9400 Variable Mode Imager without PC 63-0055-78
ImageQuant™ solution 1.4 manuals & software
set for Typhoon 9400 Variable Mode Imager
Typhoon Scan Control v3.0 for Typhoon upgrade 63-0046-27
Ettan DIGE User Manual
DeCyder Differential Analysis Software Manual
2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) technology is covered by US
patent numbers US6,043,025 and US6,127,134 and foreign equivalents and exclusively
licensed to Amersham Biosciences. CyDye: this product or portions thereof is
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5268486 and other patents pending. Some of these products may only be available to
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CyDye Fluor minimal and saturation dyes for internal research and development, but not
for any commercial purposes. A license to use the CyDye Fluor minimal and saturation
dyes for commercial purposes is subject to a separate license agreement with Amersham
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18-1164-85 AB, 2003-06 • p6
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