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Setup Gel Image Warping

Background: Why warping is important for 2D gel image analysis?

Usually, spots from the same protein appear at slightly different positions on different gels. These positional differences are the main reason for inefficiency in 'traditional' 2D gel image analysis packages.

Gel image with overlaid grid	The same gel image after warping

If you are used to classical 2D gel analysis packages, you might know those long and te-dious editing sessions for spot matches and spot boundaries that were found "automatically" by the software.
Delta2D's image warping eliminates these running differences between 2D gel images before doing spot detection or quantitative analysis using an image processing method called Warping. The result is like having perfect gels: after warping, corresponding spots have the same position.
DECODON pioneered the use of image warping in 2D gel analysis with the release of Delta2D version 1.0 in 2000.

DIGE and other multiplex methods allow you to co-migrate multiple samples on the same gel. Thereby they mitigate the problem of differing spot positions. However, as soon as an experiment contains more than one gel, gel analysis software will have to deal with different migration positions.

Here you see a combined image that is made from two gels. One is colored in orange, the other one in blue. In the dual channel image without warping it is hard to find corresponding spots and do comparisons of expression patterns.

Two gel images, overlaid into a dual channel image, not warped.	Two gel images, overlaid into a dual channel image, after warping. Differences in expression levels are clearly visible. Warping allows Delta2D to assign corresponding image positions across a whole set of images. This enables, for example, 100% Spot Matching and Image Fusion.

After the warping, the dual channel image gives valuable insight for comparing the spot patterns qualitatively. Blue spots are stronger on gel A, orange ones on gel B. Dark colors mean that spots have roughly the same intensity on both images.
But the advantages of warping go far beyond the making of dual channel images. The effect of applying image warping is as if you had made perfect 2D gels: those would have all proteins migrating exactly to the same position. And because Delta2D knows about pixel-by-pixel correspondences between images, a number of Delta2D's other core technologies are now available:

• 100% Spot Matching
  Delivers you higher statistical confidence for the analysis of expression profiles.
• Image Fusion
  Lets you combine multiple images to produce, for example, average images.
• Proteome Maps
  Use union fusion images e.g. to store protein identifications.
• Color Coding
  Analysis of Expression profiles with spot color coding combines image fusion and 100% spot matching to deliver a high-level overview of groups of proteins having similar profiles.

Defining a warping strategy

The warping for a whole experiment in Delta2D is based on pairwise warpings between gels. You do not have to produce match vectors for every possible gel image pair in your project: Given a warping between gel A and gel B, as well as a warping between gels B and C, Delta2D will automatically create an implicit warping between A and C.

Implicit gel image warping

A 'Warping Strategy' contains the definition of pair-wise warpings that Delta2D should use to construct the overall warping. It can be flat or hierarchical.
The Group Warping Strategy is suitable for most projects with replicate groups. The general guideline for setting up a warping strategy is to connect similar gels directly, and to make sure that every possible gel pair can be warped by combining a few direct warpings. See the manual section 'Defining a Warp Strategy for the Complete Project' for more information.
With the Group Warping Strategy you warp the two gels within every replicate group using the automatic warp mode. Connect the replicate groups by warping the first gel of the 'control' group (control_01) to the first gels of the '1min' and '10min' groups, again using the automatic warp mode.

Switch to the second step of the 'Workflow' window called 'Setup Gel Image Warping' and click on the 'Warp Strategy...' link to open the Delta2D Warping Strategy Manager. Alternatively you can open it via the menu: 'Gels' > 'Set Warp Strategy...'. Select the Group Warping Strategy. Automatic warping for warpings within as well as between groups is automatically taken as default setting. Press 'OK' to apply the selected strategy.

Warping Strategy Manager

You could also define the warping strategy by dragging images onto each other in the 'Warping Setup' window. Using the Warping Strategy Manager is, however, easier and faster.

Automatic warping employs the SmartVectors^TM HQ technology and delivers the result as a set of match vectors that connect corresponding spots. Always check the resulting dual channel image and if necessary improve the warping iteratively as described later in this document (step 3 'Create Direct Warpings').

The Strategy Manager overrides previous manual assignments of warping methods. So apply it after having organized a new project and be careful when applying it again later. Existing sets of match vectors are not affected.

Open the 'Warping Setup' window by clicking on the link 'Setup direct warpings' (or 'Window' > 'Warping Setup') to check whether the warping strategy has been applied correctly such that all images are connected:

Setting the warp strategy. The Warping Setup window after applying the Group Warping Strategy (Grid Layout).

As explained above, the warping strategy uses automatic warps to connect images within the same group, and to connect the first image in every group to control_01. The 'Warping Setup' reflects this: a line connecting two images means that the images should be warped directly. All the other gel image pairs can be warped by combining one or more of these warpings if each images has at least one connection to one of the other images of the project..

As a guideline, you can think of the yellow circles as showing you where you have to do work for the warping step. By creating match vectors for each of these image pairs, we allow Delta2D to warp the whole project.

Setting up a warping strategy for DIGE
and other multiplex projects

Delta2D offers a special warping strategy for DIGE and other multiplex experiments that use an internal standard. Within the same gel, we will not have to do any warping because samples are co-migrated. For warping between gels, we use the internal standard images because they always show the same internal standard sample which makes them very easy to warp.

Before you start, make sure that you have checked 'use internal standard' in project properties, and assigned gels and internal standard images.

Choose 'Warp Strategy...' from the second step ('Setup Gel Image Warping') of the 'Workflow' window to open the Delta2D Warping Strategy Manager. Select the In-Gel Standard Warping Strategy. Warp mode within gels should be set to 'Identical', Warp mode between gels to 'Automatic'. Then press OK to apply the selected strategy and close the Warp Strategy manager.

Warp mode 'Identical' means that Delta2D will take the images as-is, without doing any warping. This is suitable for images from the same gel. Of course, you can change this setting if you want to apply Delta2D's warping (e.g. if there is a minor shift between the different images).

Warp Manager showing In-Gel Standard Warping Strategy