Chapter 4
Working with Analysis Projects

The Project Manager (Figure 4.1) is the main window for creating and modifying analysis projects. It also shows the status of the gel images in your project, e.g. whether they are quantitated already, or if there are labels available.

Figure 4.1: The project manager

4.1 The Gel Pool

The gel pool is the central repository for all your gel images. Along with the images, it keeps your labels, quantitation results and match vectors. You take the gel images from the pool and group them into projects for analysis. A gel image can be part of more than one project.

Delta2D keeps the pool in a directory on your hard disk. Normally, you won't have to deal directly with the files in the pool, but rather use the Project Manager to access and organize data in the pool. It is also possible to use several independent pools. To change to another pool directory, or create a completely new pool, use Project Open. . . and click on the Change Pool... button. Now either select your preferred directory or create a new one by clicking on Create Folder. Type in the name for the new folder and confirm your input with Enter. Make sure the new folder is selected and click on OK to open it. If you confirm the following security request, the new folder will be transformed into a pool with the necessary structure, ready to hold your data.

Since the freshly created pool is empty, as next step you will be asked to create a new project. Enter the name, the author and maybe a short description in the appropriate fields and create the new project by hitting OK.

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Do not change the file structure below the pool directory and do not edit the files, otherwise Delta2D may not be able to find the data or to build up your projects. You can move or copy whole gel pools to other directories, drives, or network places. To make a backup of your data, just save the pool directory. Of course, you may read the data kept in the pool directory at any time using third party software.

Note:

With version 3.1 and again with Version 3.4 of Delta2D the data format used in the pool has changed. Opening pools created with earlier versions (< V 3.0) represents no problem, but the opposite way does not work. If it still is necessary to work with older versions on a pool which was in use with version 3.1 or newer, you can export the pool from the newer version of Delta2D in the former format. To do this, please select Pool Export As Version 3.0 . . . . Contact us for assistance to save a 3.4 pool in a format readable by Version 3.1, 3.2, or 3.3.

Adding Gel Images to the Pool

Figure 4.2: The Gel Image Manager

A small double preview of the selected image is instantly created. (In case your images folder is accessed through a slow network connection, a slow CD- or DVD drive and/or contains very large gel images this can take a while.) The left thumbnail preview shows the image in the state it is before applying any changes to it, whereas the right preview shows an instant preview of any change you apply with the buttons below: Use the button to flip the image horizontally and the button to flip it vertically. To rotate the image, use the button , and invert it with .

Figure 4.3: Image Import Dialog

Note:

The gel pool contains copies of the gel image files that were imported into it. Thus, the original data is left unchanged and you can continue to work with the gel images even if the original files are moved or deleted.

Assigning Gel Image Attributes

Assigning gel images to the corresponding gel, sample and channel is necessary for multichannel projects (e.g. DIGE setups, for details on multichannel techniques please refer to section 4.7), but can also be quite useful in traditional projects for administration of you gel images. There are two ways to do this: right away during import of the gel image (fig. 4.4) or later on in the attributes dialog (fig. 4.6).

Figure 4.4: The gel image properties dialog, available when importing images into the pool, or by right click on an image thumbnail in the Project Manager.

Each of the drop down boxes is preconfigured with reasonable values from which you can select one immediately. To create your own assignments, simply choose the second option of any of the drop down boxes, saying Add new Gel (Channel, Sample respective). In the now upcoming dialog (fig. 4.5) you can create a new item by typing in the desired name and assigning it the desired color. The newly created item is set as assigned to the current gel image and added to the selection list of the respective drop down box, thus available to be chosen for any other image.

Figure 4.5: Create a new gel image attribute, here a gel.

Figure 4.6: The gel image attributes dialog, accessable from the Project Manager, by choosing the Group Attributes. . . menu item.

Context menu:

select one or more gel images in the list, right click on (one of) the selected image(s) and assign the respective attribute in the context menu to all selected images. Using this method, you can also do the assignments of Sample and Channel without switching to the other tabs of this dialog.

Drag and drop:

again select one or more gel images in the list, click on one of them and drag it on the target gel without releasing the mouse button. If more than one image is selected, please make sure to hold down the Shift key when clicking on one of the images to "drag" them. As soon as your target gel is highlighted, you can "drop" the images by releasing the mouse button and the images are assigned to this gel.

If you want to introduce a new Gel to be assigned, please click on the button on the top right of the dialog. To remove a gel from this list that you don't need anymore, select the respective gel and click on the button on the top right.

4.2 Opening, Creating, and Removing Projects

Figure 4.7: The project manager dialog

Use Pool Projects... to open a dialog that displays all available projects. Here you can also create and remove projects.

To create a new project, press the New button in the Project Manager dialog. You will be prompted for a project name, author, and comments. You can also use the Project New... menu to create a new project. To remove a project, select it in the table and press the Remove button.

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Please note that, if you delete a project, no associated gel image, match map and quantitation information is removed from the pool. Gel images (including all associated information) can be deleted manually in the Gel Image Manager.

4.3 Gel Image Groups

Usually, the gel images of a project will come in groups. Supposed you have two samples and produce three replicate images per sample, then you would place the replicates from one sample into the same group. Grouping of replicates helps for the later calculation of the minimal, maximal, average or median expression of protein spots. Further the relative standard deviation and t-test parameter can be derived.

Creating a New Group

Figure 4.8: Creating a group

Adding Gel Images to a Group

You can now add more gel images to a group by right clicking on the table header and then choosing Add gel to group.

4.4 The Project Manager

In the project manager, every gel is represented by a thumbnail image. Drag the line between two header cells to make the thumbnail larger or smaller. You can drag the gel images to change the order of the gel images. A small icon in the header indicates whether there is a quantitation result available for the gel image. Another icon shows if there are labels attached to this gel image. As a rule, icons appear only if spots are detected or labels exist, respectivels.

You can invoke operations on a gel image or on a gel image group by using the entries in the thumbnail's context menu (see Table 4.1). Right click on a gel image thumbnail to open the context menu.

Open Dual View with choose another gel image to open the selected gel image with in the Dual Image View Move Gel Image to Group choose the group to move gel image to Add Gel Image to Group . . . add a gel image that is not used in the current project yet from the pool to the selected group Remove Gel Image from Group remove the selected gel from group Gel properties . . . shows properties of the gel and lets you add a comment Fuse all Images Create a new image, by fusing all images of your project (see section 4.8) Fuse Images in This Group Create a new image, by fusing all images of the group you choose. Quantify Gel Image . . . detect spots on the selected gel (Only applicable if no quantitation data available Transfer Spots to Gel Image Transfer the spots boundaries of the selected gel to other gel(s) (see section 4.9) Spot Color Coding uses the selected gel image as bases for a new Spot Color Coding view Collapse group Collapse all gel images of a group under the currently selected gel Remove group remove the selected group from the project Group properties . . . change name and color of group Table 4.1: The context menu in the project table header

4.5 Connecting the Gel Images by Pairwise Linkage

Everything that you can do with a pair of gel images is represented by the matrix cell, where the gel's row and column cross. Double clicking on any cell will open the Gel Image Pair View on the gel pair you have selected. Right click on the pair to get a menu of available operations (Table 4.2).

Status Icons

Figure 4.9: Details in project table

In each cell of the project manager's table, you can see the status information for the gel image pair.

On top right of the match status line which is placed on the bottom of each table cell you can see the icon if match vectors exist for this gel image pair.

The very left icon indicates the warp mode applied to this gel image pair and can look like this:

	Automatic Warp mode
	Exact Warp mode
	Global Warp mode
	Identical Warp mode and
	Implicit Warp mode.

For a more detailed description of warp modes please refer to section 5.7.

In the example project we have used the exact warp mode and the implicit warp mode only.

In the center of each cell you can see one of the following icons that provide feedback about the project's warping status:

	These two images can be warped according to the defined direct warp mode, since either no match map is needed (e.g. for warp mode identical if the images come from the same gel) or the match map contains approved match vectors.
	If the automatic warp mode is chosen, this icon means that you shall be aware of newly created non-approved match vectors. You should review these match vectors. Furthermore, the icon appears if the match map does not contain approved vectors but the warp mode (e.g. exact warp) needs a match map.
	No explicit warp mode is specified for these two images, and the implicit warping (please refer to section 5.7 for details) cannot be computed because the chain of defined warpings between these two images is not complete.
	These two images are to be warped implicitly. In contrast to the previous icon description now you have defined too many direct warpings. A so called Warping Cycle occurrs, the matching may face conflicts. (For more details on warping cycle please refer to section 5.10)

What steps result from this status?

• In pairs with the green icon a working explicit warping is defined, you can locate them instantly if you want to review or refine the warping, but no intervention is necessary.
• Image pairs with the yellow icon need a verification of the warping in the Gel Image Pair View
• The red icons indicate that your warping strategy is broken and mark the pairs affected by the problem. You can use use the Strategy Manager to redefine the warping strategy for your complete project with one click, or fix the problem manually: In most cases it suffices to change the warp mode of one of the denoted pairs: define an explicit mode in broken chains or set it to implicit in warping circles.
• In cells where you can't see one of the icons above, everything is fine.

As soon as you open a gel image pair in the Gel Image Pair View, the icon in the top left corner appears, indicating how the two colors of the false color image are assigned to the image pair.

At the very bottom of the table cell you find a bar, the quality control for matching. Depending on the situation, the coloring indicates the state and quality of the matching of spots between the two gel images:

	No quantitation data on both gel images
	Quantitation data is present on at least one gel, but no matching data is available, e.g. match vectors have been changed but the matchings are not updated yet.
	The black range of the bar represents the number of spots being present on both images and match to each other. The blue area indicates the amount of spots being present only on the image of the cell's row, the orange area those on the image of the cell's column.
	Matching is updated, but detected spots are available only on the image of the table cell's row.
	Matching is updated, but detected spots are available only on the image of the table cell's column.
	The ideal matching: every spot on the first gel image has a corresponding spot on the other image of this pair.

By pointing with the mouse cursor on such a bar for one or two seconds, a tool tip appears that includes the exact numbers.

A right-click on the table cell for a gel image pair opens a context menu with actions related to this pair of gel images, as shown in table 4.2.

Dual View opens the Dual View window and lets you edit spots, labels, and match vectors Warp Gel Images calculates the transformation for this pair of images, if a matchmap is exists or if Delta2D is allowed to employ the SmartVectors Technology using the automatic warp mode Scatter plot shows a scatter plot with the ratio of the relative spot volumes Warp Mode lets you determine the warp mode for this gel image pair (see 4.10). Status shows a detailed report about the current status of this gel pair Table 4.2: The context menu in a project table cell

4.6 Warping Strategies

Before you can create expression profiles across all the images in your project, you will have to specify enough transforms such that each image can be (directly or indirectly) connected to any other image from the project.

The warping of a complete project is done by composing pairwise transforms. These can be exact, global, automatic, implicit, or identical. Identical transforms are used for registering images from the same gel, e.g. DIGE or Multiplex experiments. Delta2D does not need a direct connection between all gel images in order to be able to warp one onto the other. There can be several intermediate steps (each of them exact, global, automatic, or identical) in-between two images. Again, this chain of intermediate steps will be used both for producing dual channel images and matching detected spots. Delta2D will always minimize the number of intermediate steps, and it will prefer manually specified warpings (exact, global, automatic with match vectors, or identical) over the completely automatic warpings.

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Note:

Please try to keep the warping chains as short as possible to reduce the number of necessary intermediate steps in implicit warpings. If implicit warping between two gel images has to be done over too many steps, small inaccuracies, which are hardly noticeable in single warpings, can sum up to bigger deviations and thus prevent small spots from matching each other.

Two extreme examples: in the One-To-All strategy the maximum of necessary steps to connect any gel with any other is two (A -> Central Image -> X), whereas in the Chained Warping strategy the number of steps for connecting the last gel image with the first one is n-1 for n gels (A -> B -> C -> ... -> X).

4.7 Different types of Experiments need different Project Setups

Standard (Single Channel) Experiments

Usually the standard experimental setup is used. For this purpose each gel of an experiment separates exactly one sample which will be stained with the same staining reagent. The gels are scanned in a single path by using only one optical channel (single channel scanning) e.g. white light scanning, fluorescent scanning OR autoradography. This results in exactly on image per gel.

DIGE Experiments Using Internal Standard

Multichannel techniques like DIGE or other multiplex techniques are based on multichannel scanning of exactly one gel.

For DIGE up to three samples can be separated simultaneously on one gel. They are covalently labeled with three different fluorescent dyes (one stain per sample) prior separation. This is possible because after the separation process the samples can be distinguished by using differential excitation and detection of the fluorescent dyes by using the corresponding multifluorescence scanners (Fuji ...). This results in exactly one image per sample but multiple images per gel (up to three samples per gel). These multiple images positionally correspond to each other. That means no further image warping will be necessary for image analysis. Because this setup is limited to exactly three samples some enhancements of this technique had been developed. For the analysis of more than three samples the so called In Gel Standard was introduced. The task of the In Gel Standard is to quantitatively link all samples although they are separated on independently prepared gels. The internal standard is a equiconcentrated mixture of all samples involved in the experiment. That means, if you are separating 4 samples A, B, C, D in one experimental setup the Standard S is a mixture of (A+B+C+D) / 4, For this experiment exactly 2 Gels have to be prepared. One gel separates S, A and B, the other one S, C and D. All spot quantities are normalized to the Standard resulting in quantities described by the formula normQ(SpotX of sampleA) = relQ(SpotX of sampleA) / relQ(SpotX of S). Since each spot from any gel is normalized to the internal standard the results are very reliable.

Delta2D and DIGE

In the Project Manager, choose from the menu Project  Project Properties to open the properties dialog for the current project and mark the Project as DIGE project by setting the tick on Use Internal Standard in the Project Properties.

Distinct from traditional setups, images from the same gel but different channels do not need to be warped to each other. Delta2D takes account of this and warps these images as identical. For a correct handling of the Multiplex-images, it is necessary to assign them to the corresponding gel, sample and channel. On how to do assignments, please refer to section 4.1.

Compared to traditional projects, Multiplex-projects are treated slightly different in quantitative analysis:

• Spots on the standard gel image are used as normalization, which means that matching spots on other gel images refer to these spots as to 100%. Due to this, spots on other gel images that have no matching spots on the standard do not appear in any representation of Expression Profiles.
• Standard gel images do not appear in the All Gel Images tab of the quantitation table and are not taken into account for statistical calculations.

In Multiplex-marked projects, on assigning gel images to a certain gel there will appear an additional radio button on the left side of the gel image's name. This radio button determines the standard image for this gel.

Another way to assign or reassign the standard image for a gel is accessible from the Project Manager: right click on the thumbnail of the gel image you want to assign and check the respective menu item in the upcoming context menu.

Other Multiplex Experiments without Internal Standard

Non DIGE multiplex techniques are also based on multichannel scanning. Here only one sample is separated per gel but differentially detected by using different kinds of staining or labeling techniques. Typical examples are the detection of protein amount (Coomassie, SyproStains or Flamingo^TMfor example) and protein synthesis (autoradiography of the same gel - only possible if the proteins were radiolabelled in vivo by using 35S Met for example). Also the complementary detection of Phosphoproteins (Diamond ProQ) or Glycoproteins (Emerald ProQ) from the same gel is possible. This results in several images per gel. Because of the sequentially applied staining techniques the gels (or scanned gel images) show typical swelling or shrinking effects which can usually be compensated by using the global warp mode. For the analysis no internal standard is used.

Delta2D and other Multiplex Experiments

In Delta2D you can analyze these experiments just like any standard project, each spot on a gel image will be normalized on the entirety of all spots on this gel image. The only difference to standard experiments is that the warp mode between different channels of one gel has to be identical or in case of minor differences caused by shrinking and swelling during the experimental handling as global.

4.8 Image Fusion

Image fusion based on image warping is one of Delta2D's outstanding features. It combines multiple gel images to one new, artificial but realistic looking composite image. You can combine all images in one group or even your entire project.

Why image fusion make your work much more efficient compared to other 2D-analysis packages

Fused images are useful in many ways:

• Obtain 100% matchings when generating a project wide spot consensus on such a proteome map, thus having definite expression profiles.
• Save time by doing spot editing and filtering only once at only one place and not on every gel of your experiment.
• Keep track of all proteins identified during long periods of experiments.
• Produce valid illustrations you will never achieve with physical procedures.
• Relativize experimental variation through several replicates in one valid representation.
• Reduce large numbers of replicate gel images to one representative.
• Condense the whole experimetns information in one representative proteome map summarizing spot identifications and expression behavoir.

How you can control the results of image fusion

Depending on the purpose, four different algorithms can be used:

Average Fusion

This algorithm averages the gray levels of corresponding pixels. A spot that is visible on only a small fraction of the gel images will be suppressed by the background in the majority of images.

This algorithm is useful for compensating statistical or experimental variation between replicates.

Union Fusion

This weighted average function places relatively high weight on dark pixels. A spot that is only present on one or a few of the images will be retained in the fused image because it is given high weight compared to the lighter pixels. Slight variations in spot positions still produce a realistic-looking spot on the fused image, and the frequency of a spot in the collection does not significantly affect the result of the fusion.

This is the most robust method and is recommended if you want to create a proteome map showing each spot appearing on any gel image used for this fusion.

Max Intensity

This algorithm prefers the near black gray values (selecting the darkest pixel of all the input images for the fused image). In the presence of saturated spots on some of the used gel images the fused image will display a combination of the input spot shapes that sometimes does not look realistic.

If you have a clean background (no artificial signals like speckles, scratches, breaks or fingerprints) and no saturated spots we recommend using this approach for the generation of proteome maps.

Min Intensity

This algorithm prefers the near white gray value (selecting the lightest pixel of all the input images for the fused image).

This method is useful if you want to visualize the minimal proteome over a whole experiment.

Figure 4.10: Image Fusion dialog

The drop down fields let you determine the steps of the fusion process in detail:

For more information about this matter you can refer to the article "Using standard positions and image fusion to create proteome maps from collections of two-dimensional gel electrophoresis images", published in Proteomics 07/2003

4.9 Transfer Spots Shapes to Other Gel Images

Especially in combination with Image Fusion this feature is quite useful: it lets you transfer spot boundaries from one gel to other gel images or even complete groups of other gel images. As an example of appliance it is a very common technique to detect spots on a union or max intensity fusion gel of all images in the project and to transfer them to all other gel images in this project. The spots are transferred following existing matchings between the images and the regions enclosed by the spot boundaries are quantified again. As a result you will have identical sets of unique matching spots on any gel image, varying only in the volumes of the single spots. Thus, a even more substantial predication of the variations in the spots becomes possible.

To transfer spots to other gel images, right click on the thumbnail of the gel in the Project Manager which contains the spots you want to transfer, and choose Transfer Spots to Gel from the context menu.

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Note:

Read more about the benefits of 100% per cent Spot Matching at www.decodon.com/Solutions/Delta2D/100_Percent_Spot_Matching.html.

4.10 Performing Analysis Jobs in the Background

Figure 4.11: The job manager

As soon as you assign the desired warp mode to the combinations of gel images in a new project, the corresponding warping jobs are created, waiting in the background until their results are required. This is the case if you e.g. open a gel pair in the Gel Image Pair View and apply the warp mode you have selected.

By default, the execution of tasks is stopped. To activate it, select Tools Start in the menu of the project manager. Now background execution is running; background jobs will automatically be created for warping gel images. The job manager allows you to control the execution of these tasks. Select Window Job Manager to open the job manager window (figure 4.11).

Use the play and stop buttons to control whether the job manager is running. The job manager shows the jobs that are currently on its task list. Jobs are executed one at a time, a progress bar shows how much of the current job has been completed. You can change the order in the task list by pressing the arrow buttons that are placed above the task list. A job can be deleted by selecting it and pressing the trash-bin button.

4.11 Spot Detection and Quantitation Options

The Spot Detection and Quantitation dialog allows you to control the detection and quantitation parameters for all gel images of your project in one place. Open it from the Project Manager by clicking on the , or by selecting from the menu Project Detection Parameters . . . .

Figure 4.12: The spot detection and quantitation dialog.

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Note:

If you are working with a project wide spot consensus please do not change these settings. Each change in this dialog will result in a redetection of spots on the respective gel image, which will screw up the 100% spot matching in your project.