Setting Up a Warping Strategy

Why you need a warp strategy

In Delta2D, you do not need warping for any available gel image pair. In general, for n gel images you need to define (n-1) image warpings to include every gelimage in a so called warp graph, while for the rest of the gel image pairs the warp mode implicit lets Delta2D combine these indirect warpings from the explicit warpings. E.g., in a project of 9 (=n) images you need warping for 8 (=n-1) out of the total of 36 (=n*(n-1)/2) gel image pairs.

Example: warp graph for 3 groups (blue, light orange, dark orange), 3 replicates each	The same warp graph as it appears in the project manager (ex – explicit warp mode)

Defining the Warp Strategy means to decide for which of the image pairs you would like to create an explicit (or direct) warping.

There are some general rules for selecting image pairs for warping:

• Always warp between the most similar gel images.
• Avoid implicit warping chains, if possible.
• Fused images generated by Delta2D will be integrated automatically by a global warp to the image the fusion gel image is based on.

For the direct warpings "global" and "exact" you need to create a match map. You can either define it manually or employ the new SmartVectors™ Technology by pressing "Find Match Vectors" in the dual view.

Important note: Warp cycles are not allowed! Make sure that for a pair of gel images with a direct warping no indirect warping is available.

Put replicates into groups

Running replicates enables for raising the statistical confidence in the results. Since in Delta2D the statistical analysis is based on the groups, make sure to move replicates coming from the same sample into one group. This will also facilitate to realize the appropriate warping strategy.
Since replicates by definition have to be similar to the highest degree, warping within groups of replicates obviously is advantageous.

Use the Warp Strategy Manager

You can choose a predefined warping strategy as provided by the Project Manager. Select between "all-to-one", "group warping", and others.
For more details about the strategy manager, please review the manual section Defining a warp strategy for the complete project.
Using the warp strategy manager you will automatically assign the appropriate warp modes to each pair of gel images. Where no direct warpings will be defined, the implicit warp mode will result in the shortest number of combinations to create a chain of warpings for each image pair. Defining a warp strategy will also guarantee that no warp cycles will appear. With warp circles you would receive annoying conflicts when expression profiles have to be built.

Having applied a warp strategy, the current status of the warp cells will be reflected by a status icon, providing an indication where you will have to be active and where Delta2D is able to automatically find the warp transformations.

The status icon can look like: everything is fine, Delta2D can warp immediately; there is no match map, needs to be defined (by hand or using the SmartVectors technology); implicit warp not yet available, work on the direct warpings in this warping chain; a warping circle exists, you have to change at least one of the warp modes to implicit.

Example: all-to-one warp graph for 3 groups (blue, light orange, dark orange), 3 replicates each	The same all-to-one warp graph as it appears in the project manager (ex - explicit warp mode)

Warp strategy for DIGE and other multiplex experiments

Using the DIGE or other multiplex experiments, you create different gel images from the same gel. Since between these images you do not face the annoying problem of differences in spot positions, they do not need warpings. Choose the warp mode "identical" for images coming from the same gel.
The "In gel standard warping strategy" automatically defines "identical" for images from the same gel, while it will propose warping between the standard images (for DIGE) only.
Important note: Make sure that your gel image properties are defined properly, inparticular insert the gel name before you apply the warping strategy. Furthermore, Delta2D will automatically identify the Cy2 gel images as the standard images to be warped among each other, if the gel image file name contains the name of this dye.

Example: warp graph for 3 DIGE gel setup (brown, ocean, violet borders). Each gel separated the standard (green) and two different samples. Alltogether 3 samples (blue, light orange, dark orange) with 2 replicates each had been separated.	The same DIGE warp graph as it appears in the project manager (id – identical; ex – explicit warping).

Checking

Warp the images very carefully. Remaining distortions effect not only the warping between the particular image pair, but it could be a part of implicit warpings, too.
Therefore we recommend to check some image pairs with implicit warping for the quality of the warped dual channel view. If you face problems in the warping of an implicit warped image pair, do not replace the implicit warp mode by a direct warping. Instead, review the warp status to see the warping chain and improve the direct warpings that are combined to this implicit warping.