Chapter 5
The Windows

5.1 Workflow

The Workflow window provides a maximum of support to analyze the project along the workflow as described in section 3.

The current status of the analysis is reflected by the Workflow, so that actions are provided or hidden if they are necessary or not yet available.

Figure 5.1: The Workflow

When you open Delta2D the first time or if you have closed your project before you have closed Delta2D in your last session and have canceled the Open Project dialog, the workflow offers a welcome screen where you can first change the location of the data in your filesystem and then open a new or existing project.

Figure 5.2: The Welcome Screen

5.2 Project Explorer

The Project Explorer (Figure 5.3) provides the most detailed overview on the project. It shows the groups, the images within the groups and if they have spots or labels, and it furthermore offers information on how the images are connected.

Figure 5.3: The Project Explorer

'Drag and drop' and 'double click' have a certain importance in the Project Explorer.

Gel Groups

To add a group right-click on Groups and choose Add  New Group.

You can use 'drag and drop' for images to move them between groups, and if you drop one image onto another, the respective Dual View will open. Double click onto an image to see it in the Dual View. If you double click on the spot set the Dual View will open with activated Spot Selection Tool.

'Drag and drop' is available for the detected spots as well: drag such a spot set onto another image or group, or drag it even to the whole project to transfer it to the respective images.

Gel Image Pairs

The current warp status is displayed by the following icons, which are also present in the Project Explorer and in the Warping Setup window:

	These two images can be warped according to the defined direct warp mode, since either no match map is needed (if warp mode is identical, e.g. if the images come from the same gel) or the match map contains only approved match vectors. You can review or refine the warping, but no intervention is necessary.
	The warp mode demands for a match map but contains no or non-approved vectors. Verify and approve or delete all non-approved match vectors and/or add match vectors.
	Automatic warp mode is chosen but not yet executed. Either open this pair in the Dual View, or open the Job Manager (see section 5.10) to start the automatic warping. After the automatic warping is executed the warp mode will be set to exact and the warp status will change to the yellow icon.
	These two images are not linked (neither direct nor indirect). Open the Warping Setup and either apply a Warping Strategy or manually add the missing direct links (please refer to section 5.5 for details). In the Project Explorer all these pairs appear in the subgroup Unlinked Pairs. Please note that with adding just one direct link many unlinked pairs will be linked.
	In contrast to the previous icon description now you have defined too many direct warpings. A so called Warping Cycle occurs, the matching may face conflicts. (For more details on warping cycle please refer to section 5.4).
	There are also too many warpings in the project, but this pair has been set to identical warp mode so that the conflict should be resolved somewhere else.
	There is an implicit warp between the two images. You can view the Dual View for this image pair.
	There is an implicit warp between the two images, but one of the pairs in the warp path has the automatic warp mode which has not yet been executed. Search for this pair and execute the automatic warping.

You can move pairs per 'drag and drop' between the Direct Warp Links group and the Unlinked Pairs group to change the pair's relation. If you double click on a pair the Dual View will open with activated Match Vector Tool.

Right click on the pair to get a menu of other available operations (Table 5.2):

Open in Dual View Opens the Dual View window and lets you edit spots, labels, and match vectors. Open Quantitation Table Opens the Quantitation Table. Delete Sets the warp mode to implicit, but does not delete the match map. Cut Copies the pair to the clipboard, so that you can paste it to the group Unlinked Pairs. Paste If the clipboard includes a pair, you can paste it into this pair group. Change Warp Mode Lets you determine the warp mode for this gel image pair (see 5.10). Scatter plot Shows a scatter plot for the image pair. Status Presents the current status of this image pair. Table 5.1: The context menu for image pairs

Gel Image Pair Status

Each pair is visualised with a status icon that represents the pair's general status (see table 5.2). You can get at any time a description of the status of a gel pair by right-clicking on it in the Project Explorer or in the Warping Setup and choosing Status in the upcoming context menu.

In the Status window the warp mode is indicated by the following icons:

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

For a more detailed description of warp modes please refer to section 5.5. In the example project we have used the automatic warp mode and the implicit warp mode only.

The status window also includes the Spot Matching Quality bar that indicates the state and quality of the spot matching between the two gel images:

	No quantitation data on both gel images
	Complete matching: every spot on the one gel image is matched to a spot on the other image of this pair. This partcilarly results from the approach using a fused image and spot transfer. See section 7.
	Quantitation data is present on at least one gel, but the matching is not up to date, e.g. match vectors have been changed.
	The black range of the bar represents the number of spots that are matched on both images. The blue area indicates the amount of unmatched spots on the one image, the orange area the unmatched spots on the other image.
	Detected spots are available on the one image only.
	Detected spots are available on the other image only.

Different Kinds of Projects

In Delta2D you distinguish between the different kinds of projects by the project's attribute Use Internal Standard in the Project Properties. Choose Project  Project Properties to open the properties dialog for the current project.

Standard (Single Channel) Experiments

DIGE Experiments Using Internal Standard

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 an 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). For this experiment at least 2 Gels have to be prepared, if no replicates are wanted. 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

%V(Spot X of sample A) = rel V(Spot X of sample A) / rel V(Spot X of S),

where rel V is the absolute Volume of the spot devided by the cumulated absolute Volume of all spots on the same image that belong to the normalization spot set. Since each spot from any gel is normalized to the same internal standard sample it is said that the results are very reliable.

Delta2D and DIGE

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 these images, it is necessary to assign them to the corresponding gel, sample and channel. On how to do assignments, please refer to section 6.2.

Compared to traditional projects, projects using an internal standard 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 projects using an internal standard, 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.

To assign a certain image as the standard image for its gel right click on the gel image in one of the windows (e.g. in the Project Explorer, the Light Table, or the Warping Setup) and click on Set as Standard Image in the upcoming context menu.

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Note:

Please note that in DIGE projects (for details on multichannel techniques please refer to section 5.2), it may happen that spots do not have quantities if you have decided to detect the spots on individual images: Spots on the standard gel image are used for normalization, i.e. each spot on non-standard images shall be devided by the matching spot on the respective standard image. If spots do not have matching spots on their respective standard their normalized spot volumes can not be calculated. Thus they do not show up in the Expression Profiles window, and in the Quantitation Tablesyou can not see their %V. To avoid this phenomenon we recommend to use the approach for getting 100% complete expression profiles as described in the standard workflow 3.

Other Multiplex Experiments without Internal Standard

Delta2D and other Multiplex Experiments

Choose Project  Project Properties to open the properties dialog for the current project and to make sure that the Project is NOT a DIGE project by removing the tick on Use Internal Standard in the Project Properties.

5.3 Light Table

The Light Table helps you to get your project organized. The layout can be either determined automatically by applying the Flow or the Column layout. Of course you can also freely move groups around.

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.

Adding a Group

Figure 5.4: Creating a group

Adding Gel Images to a Group

To change a group's or an image's name double click on the respective header and edit it.

Right-click on a group or an image to open its context menu.

Figure 5.5: The Light Table

5.4 Warping Setup

Before you can create expression profiles across all the images in your project, you need to define warping relations between the images by composing pairwise transformations. The complete set of pairwise warping relations form the Warp Graph.

This Warp Graph will be used both for producing dual channel images for every possible image pair and for building expression profiles of the spots. Delta2D does not need a direct connection between all gel images in order to be able to warp one onto the other. We only have to make sure that every gel image is included in the Warp Graph. There can be several intermediate warping steps in-between two images. With one of the predefined Warping Strategy you can minimize the number of intermediate steps.

To keep control on the already existing relations and to see where a relation is missing the Warping Setup provides a view on the warp graph.

Right-click on an image or a warp relation to get a context menu with available actions.

Figure 5.6: The Warping Setup

Warping Strategies

There is a more convenient way: Choose Gels  Set Warp Strategy. . . or right-click in the workspace in the Warping Setup and select Warp Strategy. . . from the context menu to open the Warping Strategy Manager.

This is a useful tool to automate the assignment of Direct Warp links to the gel images of a project. It takes care that no gel is left out and no warping cycle is created accidentally.

Figure 5.7: Apply complete warping strategies at once

Note:

Please note, that the Strategy Manager alters assignments done manually before by setting every warp mode according to the chosen strategy. So use it at the very early stage of a new project, and do not touch it any more later on. Of course, warp modes can be changed manually at any time; then you have to take care for the consistency of your warping strategy yourself.

• Since warping is much easier if the images are more similar to each other, we recommend to warp along the images' similarity. For this reason the Group Warping Strategy is suitable for most standard projects, while the In Gel Standard Warping Strategy is available and works best for DIGE projects.
• Warping one image on another always means that one image gets distorted, while the other (the Warping Master) remains undistorted. Since in most projects a control sample is used, it is very likely to use one of its replicates as warping master and later on as basis for the Proteome Map.
• Avoid warping cycles as they can lead to unpredictable results. A warping cycle is a chain of warpings containing possibly contradicting directions: If you have four gel images A, B, C and D, there are warpings between A-B, B-D, A-C and C-D, and you want to set the warping mode A-D to implicit, Delta2D does not know which warping chain has priority: A-B-D or A-C-D.

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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 All-to-one 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 -> ... -> N).

 Group

This will be the most frequently applied strategy. It assumes that your image groups correspond gel replicates, and that the difference within groups is smaller than between groups. Within groups, images are warped with one warp mode (default: automatic) and the first image of each other group is warped to the first image of the first group with another warp mode (default: exact).

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Figure 5.8: Group Warping Strategy

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 Chain

All images of your project are connected like one long chain in the sequence of their appearance in the project, no matter to which group they belong. This strategy is recommended, if your samples have been taken at successive points of time in your experiment.

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Figure 5.9: Chain Warping Strategy

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 Chained Group

Combination of the two above strategies, applicable in case your image groups correspond gel replicates, and the groups represent successive points of time in an experiment.

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Figure 5.10: Chained Group Warping Strategy

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 All-to-one

Here one gel image takes the role of a master and all other gel images are connected only to this one.

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Figure 5.11: All-to-one Warping Strategy

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 In Gel Standard

Warp each standard image to the first standard image. Other images are warped to the standard image from the same gel, if possible. This is the default Warping Strategy for Projects using an internal standard and hence only available if the current project marked as DIGE Project. (Please refer to section 5.2 for more information on DIGE Projects.)

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Figure 5.12: In-Gel Standard Warping Strategy

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Instead of applying a warp strategy you can define the relations manually: Just drag an image and drop it on another one. Please note that direct relations will only be created if there is not yet any relation, including indirect relations, between the two images.

5.5 Dual View

The Dual View shows a gel pair and lets you create or refine a warp transform between them. It furthermore enables to detect and review spots, preferably on the fusion image, and to define and modify spot annotations.

Figure 5.13: The Dual View

From the gel images, a dual channel image is automatically generated: one image is colored blue while the other image will be displayed in orange color. Having warped the images 5.5 blue means that a spot is only present (or much stronger) in the one gel image, while orange spots are only present (or much stronger) in the other gel image. You can now identify whole sets of spots whose expression levels vary. Shades of black are generated where both images have regions with similar intensity. ¹. You can click on the tabs at the bottom of the Dual View to switch quickly between displaying the single images, or dual channel image. You can use as well arrow left or arrow right to switch between these tabs (see figure 5.14.

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Note:

Delta2D can use any color scheme to produce a dual channel image. Unless you change it, it will be set to the default color scheme: white for background, a shade of blue for master spots, and a shade of orange for sample spots. Regions with overlapping spots are colored black. For changing the color scheme, please refer to section 5.5.

Figure 5.14: The tabs for controlling image visibility

The Dual View Menu

Export

Export Sample. . . Export the warped image. Export Dual Channel. . . Export the dual channel image. Export To Powerpoint. . . Export the current view as a slide to Powerpoint. Snapshot. . . Make a snapshot of the current view to export it.

Matches

Delete All Delete the complete match map. Import. . . Import a match map that fits to the current image pair. Export. . . Export the match map. Invert Match Vectors Invert the direction of the match vectors. Delete Selected Delete the selected match vectors only. Approve Selected Approve the selected match vectors as to be OK. Select Non-Approved Select the non-approved match vectors for approving or deleting them. Invert Selection Exchange selected against the unselected match vectors, and vice versa.

Spots

Detect Spots on [name 1] Open the quantitation dialog for the image. Detect Spots on [name 2] Open the quantitation dialog for the image. Delete Delete the spots from one of the images. Import Import a spot list that fits to the image. Export Export the spot list of the image. Export Picklists Export a list with marked and labeled spots for a certain picking device. Show Table Open the Quantitation Table for this image pair. Show Scatterplot Open the Scatterplot for this image pair. Show Hidden Spots Display hidden spots with dotted boundaries. Show Canceled Spots Display canceled spots with dotted boundaries. Background Region Change the settings for background region for the image.

Labels

Delete Delete labels from the selected gel image (master, sample, or both). Import Add labels from a file to the current set of labels on the master or sample gel. If the label file contains formatting information, you will be asked whether it should replace the present formatting. Export Export labels to a file. Formatting information will always be saved together with the label data. Move Move all labels from one gel to the other. Label positions will be adapted according to the match map. Copy Copy all labels from one gel to the other. Label positions will be adapted according to the match map. Swap Swap label sets between master and sample gel. Label positions will be adapted according to the match map. Label Selected Spots with Spot IDs   Create Labels on selected spots containing their ID. Label Selected Spots with Numbers Create Labels on selected spots containing consecutive numbers. If you need a prefix in the numbered labels, define it in Options  Delta2D   Labels. Label Unlabeled Spots with Spot IDs All spots without any label obtain a label containing their ID. Label Unlabeled Spots with Numbers Create Labels on all unlabeled spots containing consecutive numbers. If you need a prefix in the numbered labels, define it in Options  Delta2D   Labels. Translate Label Names Lets you batch change all Labelnames by providing a list with the current names in one column and the replacement names in another. Formats. . . Edit label formats. Delete scout2 data Delete data of a specific scout from all spots. Fetch scout2 data Fetch data with a specific scout only for those labels not containing this set of data. Refetch scout2 data Fetch data with a specific scout for all labels and override this specific data if already present.

Rollups

Show all Show all rollups. Hide all Hide all rollups. Expand all Expand all rollups. Collapse all Collapse all rollups. Colors Open the Colors rollup. Overlays Open the Overlays rollup. Navigator Open the Navigator rollup. Zoom Open the Zoom rollup. Expression Profiles Open the Expression Profiles rollup. 3D Spots Open the 3D Spots rollup. pI/MW Calibration Open the pI/MW Calibration rollup.

The Dual View Toolbar

Zoom out Move slider to zoom Zoom in Zoom 1:1 Fit the image into the window, such that it can be seen completely inside the window. Choose a color scheme. Show image histograms. Equalize images. Show or hide the foreground of images. Show or hide the background of images. Open a dialog with information about the warp status. Warp Warp the sample image. Disable warping operations and show images in unwarped status. Current warp mode: Select the warp mode for this sample gel image. Find Match Vectors: Apply the SmartVectors Technology to receive an automatically generated match map. Undo the last action on match vectors. Redo the last action on match vectors. Table 5.2: Buttons on the toolbar and what they do.

Note:

Toolbars can be torn off and placed anywhere on your screen by clicking on its "handle" at its beginning and dragging it to the desired place. To reattach it to the Dual View window, simply close the small window of the toolbar.

The Dual View Tool Panel

Figure 5.15: The tool panel.

Detailed descriptions of the tool buttons can be taken from table 5.3. The effect of your mouse actions depends on the tool you have activated. For example, with the Label Tool a left-click with your mouse on the images will create a new label. However, if the Zoom Tool is activated, the same mouse click will let you zoom into the images.

Match Vector Tool. With this tool you can select, delete or add match vectors that define corresponding gel positions. Spot Selection Tool. Select and mark, cancel or hide spots or exclude/include them in the normalization basis. If no spots are available on one of the displayed images, the spot detection dialog will appear. Spot Editing Tool. Add, split and fuse spots by defining spot edit markers (details in sec. 5.5). Zoom Tool. Increase the zoom level by clicking into the images, decrease the zoom level with Ctrl + click or drag a rectangle around the region of your interest. Label Tool. Create and edit labels, copy or move them to the other image. Table 5.3: Buttons on the tool panel.

The Status Bar

The status bar, located at the bottom of the Dual View window, shows some useful information about your work:

Position and zoom level

The left field of the status bar shows the coordinates of the mouse cursor in relation to the gel image. This makes it easy to evaluate the mouse position in a higher zoom level, to locate a certain spot quickly, as well as to access the size of a spot. In brackets near the coordinates, the zoom level is indicated.

Image Size, Match Vector count, Spots count

The middle field indicates the outer bounds of both images. This means the horizontal size of the broader of both images and the vertical size of the higher one. This value refers to the original images, no matter how the prescale is used. Besides, you get a quick information on how many match vectors and how many spot boundaries are present in this pair of images.

Figure 5.16: The status bar of the Dual View

Navigating in Images

With the buttons and or the slider you can zoom out (resp. in), whereas the button resets the view to the natural size of the image. With you fit the image size to the actual window size.

The zoom tool allows you to adjust more precisely the region you want to magnify. It is activated by using the tool panel in the top left area of the main window (Figure 5.15).

Press the zoom tool button to activate zoom mode. The mouse cursor will change to a magnifying glass. Click anywhere inside the image to enlarge it. Click and drag to specify a region that should be zoomed in. Clicking and dragging with the right mouse button will change the mouse cursor temporarily to act as a magnifying glass as long as the right mouse button is pressed. If you need a magnifying glass also while working with the label tool or others, it might be more convenient to use the Zoom Rollup (see section 5.5).

Configuring the Display Using Rollups

For better interpretation, e.g. you may wish to hide match vectors using the overlay rollup as described in section 5.5 (use Rollups  Overlays to open the overlays rollup).

The Colors Rollup

Open the Colors rollup using Rollups  Colors.

Move the mouse pointer over the dual channel image and watch the Colors Rollup. A small circle inside the Color Rollup points to the color that fits to the current combination of grey values. A numerical display of the intensity ratio (sample / master) is shown below the color square. Even though these values are computed only for a small pixel neighbourhood around the mouse pointer, they can serve as useful indicators for the expression ratio of a spot.

For printing or presentation purposes, Delta2D's color scheme can be changed to an arbitrary combination of colors for master, sample, background and common pixels.

Click inside the color square to change the color scheme. A menu appears, giving you the opportunity to change the used color scheme (see sec. 5.5) and to switch directly between the absolute mode (which is the standard mode) and the ratio mode (see sec. 5.5).

Figure 5.17: The Colors Rollup

The Overlays Rollup

Open the Overlays rollup using Rollups  Overlays.

Figure 5.18: The Overlays Rollup

Clicking one of the small control buttons will toggle the visibility of the respective objects between three modes: visible, non-visible, and auto-visibility (controlled by the tool activation).

The button Images Only is useful to hide all overlaid objects temporarily to get a view on the pure images, e.g. during spot editing. The overlays will reappear according to the overlay rollup settings when you release the button.

The Navigator Rollup

Open the Navigator rollup using Rollups  Navigator.

Figure 5.19: The Navigator Rollup

The Zoom Rollup

Open the Zoom rollup using Rollups  Zoom.

Figure 5.20: The Zoom Rollup

The Expression Profile Rollup

Open the Expression Profile rollup using Rollups  Expression Profile.

Figure 5.21: The Expression Profile Rollup.

The 3D Spots Rollup

• show single spots, whereas the shown region dynamically adapts to the size of the chosen spot (default setting), or show bigger regions of a fixed size
• show the spots opaque or as wire frame model (useful to make interlocking spots visible when viewing both gels in the Dual View)
• change the color of spots and background
• adapt the height scale to your needs (e.g. for very flat or very tall spots)

Choose Options. . . from the Project menu and switch to the 3D Spots tab in the section Delta2D to change the settings. Please refer to section 10.1 for more details.

Open the 3D Spots rollup using Rollups  3D Spots.

Figure 5.22: The 3D spots rollup

• Click with the left mouse button to freely rotate the spot.
• Press the Alt key or the middle mouse button and drag the 3D spot to zoom in or out.
• Click the right mouse button and move the scene inside the rollup.
• When viewing an area with multiple spots just click on a spot inside the rollup and watch the same spot being selected in the Dual View.

The pI/MW Calibration Rollup

Open the pI/MW Calibration rollup using Rollups  pI/MW Calibration.

Figure 5.23: The pI/MW Calibration rollup

Open Tools  Options in the menu or click on the Options button in the main tool bar. Click on Delta2D and then switch to the Labels tab. The Source field lets you choose from all scouts containing adequate data. Select the scout you want to use for the estimation and specify in the fields below which data fields are to be interpreted as pI and MW values.

Example You know the pI/MW values for 4 points on your gel and want to be able to see them for other interesting spots or regions. Here is how to proceed:

• If not already existing, place a label on every point you know the pI and MW of. The label can but needs not be connected to a spot on the gel. The names of the labels are of no importance for this purpose; they can be e.g. simple consecutive numbers.
• Right click on the first label and choose from the context menu Edit scout data  Physicochemical properties, a new dialog shows.
• Insert the values for pI and MW in the respective data fields.
• Close this dialog with OK.
• Repeat the last three steps for every label you know the pI and MW for.
• Now open the Options dialog (Project  Options) and switch to the Labeling tab in the section Delta2D.
• On the bottom of the left side you see three drop-down boxes. Click on the first one and make sure Physiochemical properties is selected.
• Click on the second drop-down box and make sure Isoelectric point is selected.
• Click on the third box and make sure Molecular Weight is selected.
• Close this dialog with OK

Now you can open the pI/MW Calibration rollup and just read the pI/MW for any region on this gel you are pointing to.

Figure 5.24: Setting the data source for pI/MW-Calibration

Controlling Background Display

Try it: Press the Show/Hide Background button to switch background visibility on and off. You can use the tabs at the bottom of the Dual View window to switch between the gel images and the dual channel image. Often the dual channel image without background is clearer than the complete dual channel image (see figure 5.25).

Figure 5.25: A dual channel image with background switched on and off.

Figure 5.26: Visual background settings

Show visual background region in spot detection dialog

means that this setting will be handed over to the Spot detection dialog. It can be seen, when you open the Spot detection dialog, but will not be applied to quantitative data before you start a new quantitation.

Overwrite visual background region after spot detection

means the opposite way of communication: if you change this parameter in the spot detection dialog, it will be changed here as well.

The purpose of these options is to keep the visual background region in sync with the technical background region, thus the default setting for both options is checked. Please note that the visual background is linked to the background detection controlled by the quantitation dialog. With the default settings it has influence on spot quantity.

The Histograms Dialog

Delta2D allows you to compensate for differences in brightness and contrast between master and sample gel images by adjusting the image histograms. This can result in production of clearer dual channel images. Histogram adjustments are controlled with the Histograms dialog, illustrated in figure 5.27.

Figure 5.27: The histograms dialog.

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Note:

Histogram adjustment is saved individually for each image. It affects the generation of dual channel images and the representation of images in the Gel Regions View, but leaves the spot quantities unchanged. It is meant to enhance the view of the gel images without changing quantitative data. This is in accordance with Delta2D's principle of leaving original data unchanged as far as possible.

Histograms or Amplitude Rescaling?

Amplitude rescale is a standard approach in image processing, which rescales the effectively used range of grey values in an image to the maximum usable range. For example: if you have an 8-bit image (which can define one of 256 grey values for each pixel, and let 0 being black and 256 being white), where the darkest pixel has the grey value 40 (dark grey) and the lightest 180 (light grey; quite a grey and shallow looking image), amplitude rescale represents the value of 40 as 0 (perfect black), 180 as 256 (perfect white) and the intermediate grey values rescaled respectively. Thus you have a much more plastic and vivid representation of your image without having altered the information contained. Depending on your gel images, the effect of amplitude rescale you can observe can vary in a wide range: the enhancement can be either hardly visible if your images already use a wide range of grey values, or it can even make spots visible you could not see before if your images use only a small bandwidth of grey values.

But of course this approach has a drawback as well: loading and displaying images in the Dual View takes slightly more time, especially if according to your zoom settings only a part of the image would be loaded, because for amplitude rescale the complete image has to be analyzed. Thus, the recommended setting depends on your gel images: if the effect of amplitude rescale is hardly visible, you can switch it off (please refer to section 10.1 on how to do so) to load images faster; if your images are generally more flat and poor in contrast, amplitude rescale can be a great help.

Amplitude rescale is nothing more than a rough pre-enhancement, which can be sufficient in many cases, but cannot take into account problems with artificial signals like speckles or gel breaks. This evokes the necessity of additional fine tuning the enhancement process. Histogram adjusting gives you control of the enhancement process. You can change the rescale settings smoothly while watching how the representation alters with your changes. But you can also let Delta2D adjust the histogram settings of an image automatically in consideration of another images contrast situation to make them both better comparable.

Automatic Histogram Equalization

Manual Histogram Equalization

The current grey scale histogram of both images of the Dual View is displayed in the dialog. The histogram display shows you how many pixels are contained at each grey scale level in the corresponding images.

To achieve optimal equalization please proceed as follows:

• Move the sliders of each image until the contrast settings fullfill your needs.
• Press the Equalize button and look at the vertical slider. If the vertical slider indicates that one histogram is higher weighted compared to the other move the left slider of that histogram a little to the right and press the equalize button again.
• Do this iteratively until the vertical slider is located exactly in the center.

If done that way you have perfectly equalized images in the dual view.

Manually Adjusting the Balance Between Two Images

Modifying the Histogram for a Single Image

Manipulating the slider positions allows you to suppress image regions containing pixel values outside a specified range from the display.

Managing the Histograms Dialog

Lock Dual Channel

Normally, if you make adjustments to the histogram of an individual image, the dynamic image display will change to show only that image. If you want to be able to watch the effect on the dual channel image while you make the adjustments, check the Lock dual channel box..

Resize the dialog

Enlarging the dialog itself may give you better control over fine histogram adjustments, since the histograms and image display will be scaled up to fit in a larger dialog.

Apply or discarding changes

Clicking the dialog's OK button will apply the changes to the images displayed in Delta2D's main frame. Clicking the dialog's Reset button will reset all the histogram values to the values that existed at the time the dialog was invoked. Clicking the Cancel button will dispose of the dialog without any changes being applied.

About the Histogram Adjustment Process

1. if the pixel is brighter than a given threshold, make it completely white
2. if the pixel is darker than another threshold, make it completely black
3. otherwise apply a linear change to the grey-level of the pixel

Adjusting histograms is advisable when your image does not use the entire dynamic range (i.e. there is no bright white or no black in it) or if there is a homogeneous background.

You may also wish to keep the following points in mind when making adjustments to the image histograms:

• Most importantly, remember that adjustments to the image histograms affect only the visual presentation of your gel images – there is no affect on the spot detection and quantitation process.
• If you have the image background subtraction feature activated, histogram adjustments will be made to the images after the background has been subtracted.
• If you have enabled amplitude rescaling, both gel images will already utilize the available range of grey scale values. You only need to make changes to the histograms if the grey levels are different in the two images, or if you want to suppress lighter or darker regions of the images from the display.

See section 10.1 for global options for image preparation.

Using Colors: The Color Schemes Dialog

The Color Schemes dialog can be invoked from the Images menu, using the Images  Color schemes. . . menu entry. Alternatively, you can click directly on the Color Schemes button in the tool bar.

Figure 5.28: The color schemes dialog.

Display Modes and Color Schemes

The currently selected color scheme is presented in the center of the Color Schemes dialog (figure 5.29).

Figure 5.29: The color schemes display.

• Top-left corner – The color used to display sample spots i.e. spots appearing exclusively in the sample gel.
• Top-right corner – The color used to display regions where spots overlap.
• Bottom-right corner – The color used to display master spots i.e. spots appearing exclusively on the master gel image.
• Bottom-left corner – The color used to display regions of image background.

If you are editing the scheme for ratio mode (see figure 5.30), you can choose two additional colors for highlighting points with only a relatively small ratio between the sample and master spot levels. Those colors are displayed in the middle of the top edge and of the right edge respectively. The ratio mode was implemented for a more fine grained representation of expression changes especially for faint spots.

Using Predefined Color Schemes

Figure 5.31: Some predefined color schemes

Defining Your Own Color Schemes

Create a New Scheme To create a new scheme, click on the New Color Scheme icon . A new scheme will be created with the same colors and a similar name to the currently selected scheme.

Select New Colors for the Scheme You can now configure the colors to use in your new scheme. First, click on the corner of the color scheme display corresponding to the color you want to edit. For example, if you want to define a new color for sample spots, click on the top left corner of the color scheme display. You can then choose a new color to use for highlighting the selected image feature. There are three methods available for doing this, accessible at the bottom of the Color Schemes dialog. The three color controls are described individually below.

Color swatches

This is the simplest color control to use. Simply select the color you want to use from the palette of available colors.

Hue-saturation-brightness (HSB) control

Using this tab, you can control the hue, saturation and brightness of a color separately. Select which of the three values you want to change by using the HSB radio buttons, and change the value by using the slider or by entering a value directly into the text field provided.

Red-green-blue (RGB) control

This control panel allows you to configure the levels of red, green and blue that are combined to produce the desired color. You can set these levels using the sliders provided, or by typing a value between 0 and 255 directly into the corresponding entry field.

It is recommended that you choose strongly contrasting colors for master spots, sample spots and the spot overlap, and an unobtrusive color for the image background, to help you to visualize the differences between two gel images quickly and clearly.

Renaming a Color Scheme

You can not rename one of Delta2D's predefined color schemes.

Deleting a Color Scheme

You can not delete one of Delta2D's predefined color schemes.

Using Ratio Mode

Ratio mode works best with images that have a low background level. Therefore you should switch off background using the layer control and adjust the histograms, if necessary. Select Images  Ratio mode to activate ratio mode display.

Figure 5.32: A gel region shown in ratio mode.

Warping Gel Images

When you hold two similar gel images next to each other, you may have the impression that the spot patterns on one gel are more or less a distorted version of the patterns on the other. The process of distorting (or "un-distorting") images is called warping. Delta2D's warping algorithms help you to generate dual channel images on which corresponding spots are perfectly overlaid. In the dual channel image, differences in protein expression levels can then be easily recognized. The same algorithm that is used in producing dual channel images will be used in the subsequent quantitation step to obtain accurate and reliable spot matching information in the Quantitation Table.

Figure 5.34: A region of the dual channel image, before and after exact warp. Corresponding spots are overlaid exactly, allowing for easy identification of spots with changed expression level.

Here is a more detailed description of the warp modes:

[Identical Warp mode] This is just no warping at all. Identical transforms can be used for registering images that are from the same gel but display different samples or multiple aspects of a sample.

[Global Warp mode] Compensates global gel distortions such as growing or shrinking, rotation, stretching a special smooth transformation can be used. Set a few match vectors then use global to see more correspondences. Global warp is a good start for setting more and more match vectors by hand. It is almost never suitable for producing the final dual channel image because there are local distortions as well. As a result you will see that the match vectors are shortened substantially but not set to zero, as the exact warp mode does (see figure 5.35).

After the global warp you will see more corresponding spot patterns because the sample image is better aligned to the master image. Fix some more matches. You do not have to fix every correspondence you see, assigning a single spot pair is usually sufficient to align the region around it. Since all vectors are weighted similarly for the global transform outliers can be recognized very easily. That's why the global warp is often used for finding warping errors.

[Exact Warp mode] All spots that are connected by a match vector will be perfectly overlaid after warping. Other spots will be warped according to match vectors in their neighborhood.

The difference to the global warp can be seen in figure 5.36.

[Automatic Warp mode] Let Delta2D try to automatically find match vectors by analyzing similarities in the gel images using the SmartVectors^TM Technology and apply the set of non-approved match vectors to an exact warp. If match vectors are present they will be used to guide the automatic warping process so that you can use the automatic warp mode iteratively and in combination with manually defined match vectors (see 5.5).

As with exact warping, spots that are connected by a match vector will be perfectly overlaid. Start the automatic warping by starting the Job Manager, or press Find Match Vectors, or just press the Warp button if no match vectors exist yet. When the process has ended the warp mode will is set to exact warp to avoid endless loops. You shall always review the result of automatic warping.

Read more about SmartVectors^TM at www.decodon.com/Support/Howto/SmartVectors.html

[Implicit Warp mode] Warp the images by a combination of explicit pairwise transformations (these can be exact, global, automatic, or identical) that connects them. Example: Image B has a valid warping to image A, image C is also connected to image A, then image C can be compared to B using implicitly the existing warpings: C  A  B. You will usually have implicit warps for most of the gel pairs in your project.

Warp images with the defined warp mode by pressing the warp button , unwarp by pressing the unwarp button . If a set of match vectors (the match map) exists, it will be applied. If no match vectors exist but automatic warp is chosen, new match vectors will be found. Otherwise the warp button is deactivated since either a warp mode is chosen that does not demand for a match map or you have chosen a warp mode that demands for a match map, but there is no. Please read on about how to define match vectors in section 5.5.

Figure 5.35: A region of the dual channel image, before and after global warp. Corresponding spots are not overlaid exactly, but much closer than in the original image. Further correspondences can be identified more easily.

Add More Match Vectors if necessary

Figure 5.36: An image region with well-aligned spots after exact warp (left image) in comparison to the same region after global warp (right image).

Note:

Existing match maps will not be used if the identical or the implicit warp mode has been chosen. However, an existing match map will not be deleted by just changing the warp mode. Switching the warp mode back to global, exact or automatic will let Delta2D use the match map for warping.

Saving the Warped Image

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Note:

Images exported in warped state can be used for documentation purposes only. They are not suitable for further quantitative analysis of any kind (reimported in Delta2D or imported in other software). Warping alters the complete image, which affects spot size as well. According to Delta2D's principle to leave original data unchanged, all quantitative analysis is done on the original, unwarped images.

Setting Match Vectors

Before you start to set match vectors, make sure that match vector tool is activated by clicking in the tool panel.

Global options for match vectors can be defined in the Options dialog (see section 10.1 for more details).

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Note:

With version 3.4 we have introduced Undo and Redo for match vector operations. Thus you can try setting a couple of match vectors, study the resulting dual channel image and go back to previous match maps if you like.

Figure 5.37: Setting match vectors.

• Click on a spot in the sample image. It is marked by a solid circle.
• Click again to set the corresponding position in the master image. It is marked by a solid circle.

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Note:

It is important to draw all match vectors from sample (orange) to master image (blue).

Note:

In order to use automatic snapping of match vectors the option Match Vector Snap in the Options dialog (Section 10.1) must be enabled.

Questions and Answers About Warping

• How do I know which warping mode is the most adequate for my gel pair?
• By default, Delta2D uses implicit warp mode for any gel image pair. This is a reasonable choice because in the end most of the image pairs will be connected using implicit warps. Usually you have to decide between two warp modes that you can assign for image pairs in the Project Explorer or the Warping Setup:
  • Between images from the same sample, choose automatic warp mode.
  • If you have multiple images per gel, choose identic warp mode between all images that were made from the same gel. If there were some experimental (except scanner reconfiguration and scanning) steps between recording of the images, try global, or, if this does not work, exact warp.

    Using a Warping Strategy is highly recommended. Please see section 5.4 for more details.

    When you are ready with warping, the Project Explorer} and the Warping Setup should show only green symbols for the directly linked image pairs. Please refer to section 5.2 for information about how to deal with yellow or red symbols.

• I only want to do quantitative analysis, do I need to warp the images?
• Yes. And no. If you just want to obtain quantitative data for single gel images without comparing them to each other or for images from the same gel, this is possible without match maps. But to compare multiple gels Delta2D demands for warping to compute a fused image where you can detect the project wide consensus spot pattern and to transfer this spot pattern back to the appropriate positions on all images. Furthermore, scatter plots require spot correspondences.
• Do I have to create a match map for every gel image pair in my project?
• No, Delta2D can combine warp transformations to connect two images indirectly via a set of directly linked images. Thus, Delta2D assists to find a good warping strategy for your project (see section 5.4.
• What if one gel image is substantially rotated or shifted with respect to the other?
• Assign a few correspondences that you can identify reliably. Then use Warp  Global Warp to eliminate global distortions. The global warping will bring similar spot patterns closer together, compensating for global distortions such as shifts, minor rotations, or differences in scaling. For a shift, already one single match vector is enough. If the rotation is > 90o, please rotate the respective image in the gel image properties dialogue (available from its context menu).
• What if initial match vectors are hard to find?
• With highly dissimilar gel images it is sometimes hard to find the first spot correspondences. Assign as many correspondences as you can identify reliably. Then use Warp  Global Warp to eliminate global distortions, again bringing similar patterns closer together.
• When or why use global warp?
• Use the global warp early in the matching process, when you are not sure about further correspondences. Global warp is more robust with respect to wrong correspondences – one wrong match vector will not distort your image too much. Nevertheless, after the global warp, further correspondences will be easier to recognize.
• Does warping affect the quantitation process in any way?
• Not at all, spot detection and quantitation is done using the original images.

The Spot Detection and Quantitation Dialog

spot detection

– identification of image segments that are occupied by spots

spot quantitation

– summing up the grey values of the pixels belonging to each spot. Background is subtracted, and calibration curves (if available) are adapted. Normalized volumes are provided in the Quantitation Tables while raw volumes can be reviewed as well.

spot matching

– assembling single spot quantities to expression profiles. For transferred spots this results in Complete Expression Profiles.

Spot detection is done automatically, controlled by a few parameters that are proposed by Delta2D but can be changed by the user. In Delta2D, any "spot painting" or "cutting" by hand is obsolete. However, you can edit the spot pattern by cancelling, splitting or joining, or by adding new spots (see section 5.5 for details). Starting the spot detection for a single image (probably on the fused image) is easy: Right click on the respective image (probably the fused image) in one of the windows (e.g. in the Project Explorer, the Light Table, or the Warping Setup) and select Detect spots. . . from the context menu. Delta2D presents the Quantitation Dialog (figure 5.38) to set or confirm the settings before Quantitation itself is done.

To start the spot detection in the Dual View select Spots  Detect Spots on name of your gel image. . . . If there are no quantitations available, clicking on the Segments tool icon in the tool panel will also open the Quantitation Dialog.

The Quantitation dialog comes with a proposal for three numerical parameters. The numbers are derived directly from the images and should lead to reasonable results. However, you can change the parameters according to your individual preferences. Having changed the parameters proposed by Delta2D, you can restore the proposal again by using the feature Parameter estimation. Simply click on . Please refer to section 5.5 for a detailed description of each of the parameters.

The set of the parameters that have been used for quantitation is saved within the *.qnt files which contain the quantitation information of a 2D gel image. You can load a parameter set from a previously exported quantitation file by loading the corresponding *.qnt file in this dialog. Parameter sets can be saved and loaded using the buttons in the top right panel.

Figure 5.38: The quantitation dialog.

Quantitation is always done using the original unwarped images while warping and histogram adjustment have no effect on the results. The background for a spot is computed and subtracted automatically, it is the very same background that is switched on and off using the layer panel.

When the spot detection process is finished, spot boundaries will be shown in the main window of the Dual View. The spot boundaries for the respective gel image are overlaid on the image, placed on a separate layer (one spot layer per gel image). These layers can be switched on and off, just like the image layers, using the overlays rollup. Select Rollups  Overlays to open the rollup.

Spot centers are marked by points. The center is located where a spot cutter would obtain the maximal protein amount.

Spot Detection Parameters

Configuring the Local Background Region The local background region refers to the radius (in pixels) of the region used for local background determination in the quantitation step. This option controls the computation of background quantities (influencing spot quantities and ratios). Lower values result subtraction of more background from the spots volume, especially for large spots. Reasonable values should be 1.5 to 2.0 times the diameter of the largest spot in the image.

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Note:

This option also effects the snap to spot feature. If this parameter is set to a value that is much too high, it happens that snap to spot gets difficulties to differentiate between adjacent spots.

Configuring the Average Spot Size Specifying the average size of spots in your gel images enables Delta2D to separate overlapping spots more accurately, as well as to distinguish spots from the image background. The value specified refers to the radius of an average spot, in pixels. Higher values will decrease noise sensitivity. Use lower values to separate spot clusters better and to detect very small spots.

Note that you can get an idea of the size of the spots in your image by looking at Delta2D's status bar, at the bottom of the main frame. The status bar displays information about the current pixel position of the mouse pointer within a gel image, so you can see how large your spots are by moving the mouse over the spots and observing the number of pixels covered directly.

Figure 5.39: The cursor position in pixel count in the Status bar

Configuring the Weak Spot Sensitivity This parameter allows you to control how strictly Delta2D discriminates between spots and the small signals. Being able to configure this sensitivity is useful when you have gel images containing very weak spots. Just type a percentage value directly into the text entry field corresponding to either the master or sample gel image.

Specifying a higher value will result in Delta2D detecting spots with weak intensity more reliably. Specifying a lower value will mean that more noisy background artifacts in the images will be suppressed successfully.

Usually, a value between 5 and 20 % is suitable.

Saving and Loading Sets of Parameters Delta2D saves the parameters used for detection together with the detected spots for each gel image individually. Additionally, Delta2D lets you export and import your parameters to and from files, e.g. for exchange with other Delta2D users, or if you want to try out different settings but want to keep a special one. To save the current set of options to a file, click on the Save icon . A dialog will appear, enabling you to specify a file to save your options to.

To load previously saved parameters, click on the Load icon . A dialog will appear, enabling you to choose a file from which to load a set of parameters.

Spot Detection and Quantitation Parameters for All Images 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 by selecting Gels  Detection Parameters. . . . _________________________________________________________________________________________________________________________________________________________

Note:

This dialog is useful if you detect spots on the different images individually. Please note that the recommended workflow for getting complete expression profiles demands for spot detection on a fused image only since the resulting spot pattern will then be transferred to the other images. Asynchronous spot patterns on the different images as they come from individual spot detections cause difficulties that can be avoided.

If you are working with a fused image and spot transfer 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.

For a more detailed description of the single parameters please refer to section 5.5.

Spot Shapes: Pixel Based or Modeled

For different reasons, be it that, due to a low resolution of your image, the spot outlines look to rough, be it for purposes of printing or presenting results, or simply for a better overview, a smoother appearance of the spots its often preferred. Delta2D includes the option to model spot boundaries within the process of the spot detection. Simply check the box Create Modeled Spots when defining the parameters for spot detection and quantitation. (Fig. 5.41).

Figure 5.41: The same region with pixel based and model based spots.

Note:

The spot boundaries define the relevant area for spot quantitation. Therefore, whether you decide for modeled spots or not, the spot boundaries you face determine the quantities for the spots on your gel images. To achieve comparable spot quantities for your analysis, we strongly recommend to decide for one type of spot shapes for the entire experiment.

You have access to the option in the dialog for the spot detection parameters only. The option will be used for the next spot detection, existing spots on other gel images will not be affected. For changing the spot shape a redetection by using the altered parameter is necessary. "Keep attributes" preserves already done classifications like hidden, canceled, exclude from normalization, etc..

Spot Editing

Figure 5.42: Edit spots

Adding a Spot To add a spot, click on the Spot Editing Tool in the tool bar of the Dual View. Now click on the position in your gel image where the spot should be detected, trying to hit the darkest point of the aspired spot. Where you clicked, a "+" will appear and according to this manually added marker a new spot will be detected instantly. If the result is not satisfying, you can either move the marker by dragging it, or you can remove the marker by right-clicking on it and set a new marker somewhere else. Your manually added spots will look and behave exactly like the automatically detected spots but still keep their marker as manually added, visible when switched to the Spot editing tool. Thus reproducibility is granted and at any time you have the possibility to edit them again. In some cases it can be necessary that you drag a spot marker instead of setting it by a click. The dragged line markers have some influence on the spot shape and help for the correct detection of by gel breaks separated by spots.

Splitting a Spot in Two To split a detected spot in two parts, simply override the detected spot with two manually set detection markers: select the edit spots tool as described above. Now click on the two sections of the spot you want to divide, trying to hit the centers of the aspired spots.

Joining two Spots If a spot was detected as two spots wrongly, you can easily join the two spots: switch to the edit spots tool, and drag a line from one to the other half of the aspired spot. Delta2D will join the two spots touched by the line.

Removing a Manually Edited Spot To remove an edited spot, choose the edit spots tool as described above. Now right click on the marker you want to remove.

Spot Quantitation and Matching

Quantitation data can be saved as a complete set of data (spot boundaries and quantities for the whole gel image) using the Spots  Export  menu.

Since Delta2D includes image warping (introduced into two-dimensional electrophoresis gel image analysis by DECODON in the year 2000), spot matching is very reliable, even with individual spot detection for each gel image. In traditional packages for the analysis of 2D gel images, where spot matching is based on spot patterns rather then spot positions, these steps are error-prone and require extensive manual corrections. In Delta2D spot matching is done automatically since after warping corresponding spots already have the same position throughout the whole experiment.

With its intuitive and modern approach, Delta2D tries to automate the analysis as far as possible, leading faster to results you can rely on.

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Note:

Based on image warping and image fusion, DECODON has introduced complete expression profiles to avoid missing values in the Quantitation Table and the resulting problems during statistical analysis. To receive complete expression profiles, create a Proteome Map make an union fused image out of the whole set of images in your experiment(at least out of one representative image of every group of replicates). Then do the spot detection on the resulting Proteome Map only and transfer the spots to the original images. Please refer to sections 7 for details.

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

5.6 Quantitation Table

Delta2D displays quantitative data in flexible tabular views (see figure 5.43) that fit your analysis needs. Table rows can be filtered and sorted by numerical and non-numerical columns, making it easy to identify relevant sets of spots. The table display is always synchronized with the spot boundaries on the Dual View, so you can go from image to data and back again with ease.

Figure 5.43: The quantitation table

Statistics tables

include statistical values for each expression profile with respect to the groups plus data comparing the group values. This is the first table you get to see when opening the Quantitation Table window.

Multiple gel image tables

basically have the same structure as the single gel image tables, except that each row in the table represents the expression profile of matched spots. All attributes appear for each gel image. The column headers are color coded to make it easy to see from which gel the data in a certain column was taken.

Single gel image tables

show the relevant spot attributes for a single gel image. Each row represents one spot.

The Quantitation Table can be opened via the menu Window  Quantitation Table. It includes two tabs: a Multiple gel image table for all images with spots, and a Statistics tables for the whole project. For an image pair a Multiple gel table can be opened by choosing Spots  Show Table in the Dual View.

Quantitation Tables are also available by clicking on the table button . The type of table depends on what has been selected: A Single gel image table or Multiple gel image table if the selection includes a number of images, a Statistics tables if only complete groups (at least two) have been selected.

A variety of attributes is available in the different tables, some of them being hidden by default to reduce the tables' complexity. The attributes that are available in the Statistics tables are described in table 5.6. To change the different attributes' visibility, please refer to section 5.6.

The Quantitation Table Menu

Export

Export Export the visible data range as .csv (comma seperated values) file. Export to Excel Open Excel which will automatically load the visible data range. Generate Report in Excel Excel will open with some analysis features, available for Multiple gel image tables only. Export Pick Lists Export a list with marked and labeled spots for a certain picking device.

Edit

Select All Select the whole visible data range. Invert Selection Invert the current selection status. Complete Row Selection Expand the selection to the complete profiles of the selected spots. Copy Selected Rows Copy selected rows into clipboard.

Mark

Hide

Cancel

Normalization

Filter

Columns

Labels

Find Label Search the different label columns for a string. Label Selected Spots with Spot IDs   Create Labels on selected spots containing their ID. Label Selected Spots with Numbers Create Labels on selected spots containing consecutive numbers. If you need a prefix in the numbered labels, define it in Options  Delta2D   Labels. Label Unlabeled Spots with Spot IDs All spots without any label obtain a label containing their ID. Label Unlabeled Spots with Numbers Create Labels on all unlabeled spots containing consecutive numbers. If you need a prefix in the numbered labels, define it in Options  Delta2D   Labels. Translate Labels Lets you batch change all Labelnames by providing a list with the current names in one column and the replacement names in another.

The Quantitation Table Toolbar

Changing the Table Layout

Table Properties

A quick and effective way to customize the Quantitation Tablesis to open the Properties dialog of the Quantitation Table by clicking . In the upcoming dialog you can define the visibility of images and spot attributes in the table.

Figure 5.44: The properties dialog of the Quantitation Table

Ratio Master

Here you define to which image the ratio of relative spot volumes in Multiple gel image tables refer to.

Visible

Check the gel images you want to see in your table. Visibility also applies to the Gel Regions View

Choose one of the following buttons to set multiple attributes at once:

All Images

Set all images as visible.

All Columns

Set all columns for all gel images as visible.

5.7 Gel Image Regions

Figure 5.45: Same region of four different gel images

When you have opened a Dual View window, its displayed area will determine the segment shown in the Gel Image Regions window.

5.8 Expression Profiles

Figure 5.46: The expression profiles window

Simply select interesting spots in the Quantitation Table or in the Dual View. Mark the spots in the Dual View by clicking right on one of the selected spots and check the box named Mark spot, or in the Quantitation Table by selecting the menu item Mark  Mark Selected Rows. Now you can open the Expression Profiles Window by choosing the menu item Window  Expression Profiles.

You can change the size and arrangement of the barcharts with the controls on top of the window. For labeled spots each barchart shows the label in its title. Right clicking on a single barchart provides a context menu showing the spot's IDs and the opportunity to change the mark status. Unmark a barchart to exclude it from this window.

Furthermore, you can change the design of the graphs and the data shown in the menu item View:

Show Group Bars Collapsed

Combine all single bars of gel images of one group to one single bar for the whole group.

Show Mean Values

Show the mean values for each group.

Show Standard Deviation

Show the standard deviation for each group.

Connect Mean Values

Change the representation of values by bars to a line which connects the mean values, thus signaling the fold change of this spot.

Show Axis

Show a scale on the left border of each graph plus axis to make it easier to read the volume of each spot.

As in any other part of Delta2D, selection of spots is synchronized between windows.

5.9 Color Coding

Color coding for spots lets Delta2D display a gel image (or proteome map) with spots colored according to theirprofiles. For an example, see figure 5.47: are colored by the following scheme: Spots that are increased in sample 1, and in no other sample are shown in red, green is for spots that are increased in sample 2 etc. Yellow is for spots that are increased in samples 1 and 2 etc.

Figure 5.47: A Region with Colored Spots. The color of a spot indicates on which sample(s) it is increased.

Subsets

The subset of gel images on which a spot occurs is crucial for the color it will be represented with.

Min/Max

The spots are colored by the gel on which they have their maximum or minimum volume.

Switch to the tab containing the options for the type of color coding you want to achieve.

Color Coding by Subsets

To add a new match subset, click on the button. This will add a new empty subset, which you can configure by clicking on the boxes in the column. Note that if the empty subset already appears in the table, clicking the button will have no effect.

To remove a selected subset, click on the button. You can select a subset for deletion by clicking on its column header.

To add all possible subsets, click on the button. Afterwards, the table will contain one column for each possible combination of matches across the gel images.

To delete all existing subsets, click on the button.

Figure 5.48: Choose Colors and Master Image

Color Coding by Min/Max

Select the characteristic that you want to highlight. Each spot will be assigned the color of the group that most strongly or weakly represents that characteristic.

Example: Color Coding Spots by Subsets

Step 1: Detect Spots

Step 2: Show a Subset of Spots on Each Image

Step 3: Choose Colors and Master Image

Step 4: Adjust the Display

Color Coding Spots by Intensity

Exporting the Color Coded Display

5.10 Job Manager

Besides the usual procedure of doing the warping one by one by yourself, you can let Delta2D do automatic warpings for image pairs in the background while you continue to work on other things, e.g. editing labels on another gel image.

Figure 5.49: The job manager

Select Window  Job Manager to open the Job Manager window (figure 5.49). By default, the execution of tasks is stopped. To activate it, press the play button . Now background execution is running; background jobs will automatically be executed for warping gel image pairs. The Job Manager allows you to control the execution of these tasks.

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. Only one job is executed 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.

5.11 Analysis

Delta2D provides advanced multivariate statistics in the analysis of 2D gels, including:

• Heat map display of expression profiles
• Various methods of clustering
• Principal Components Analysis (PCA)
• T-tests with optional resampling and control of false discovery rate
• Analysis of Variance (ANOVA)
• Template matching for expression profiles
• Some non-parametric tests e.g. Kruskall/Wallis

Getting a High Level Overview of Expression Data - Heat Maps

Figure 5.50: A Heat Map

Let us make a heat map:

• Open the Demonstration project in Delta2D.
• Open the Quantitation Table (Window  Quantitation Table), make sure the Statistics Table is selected.
• Hide the quantitative data for the fused image: Click on , uncheck the checkbox next to Fused Image, press OK (see section 5.6 for changing the tables and/or images visibility).
• Press the Analyze button in the top left of the Statistics Table (fig. 5.51). A new analysis window is opened, containing the current expression profiles in a heat map display.
• If you want to see more rows at once, you can use Display  Set Element Size and select 20 by 5.

Clustering Images: What Image Groups or Classes Are There?

Clustering methods can group expression profiles and gel images by similarity. This can be very useful for getting an overview of all expression profiles before proceeding with more detailed analysises. Clustering of gel images can also be used to detect outliers, and to identify structures in the experiment. Ideally, the cluster composition will reflect the structure of the experiment, e.g. replicates and images from the same sample should have similar expression levels and thus end up in the same cluster.

Figure 5.52: In this clustering you see an experiment with control (C1, C2, C4, C5) and treated (T1, T2, T3, T4) samples, made in triplicates. The clustering rediscovers the experimental setup, i.e. gel images with similar samples share a cluster. A sample forming a separate cluster would indicate an outlier for which closer inspection is advisable. Made using Pearson correlation as the similarity measure between images.

• Press the HCL button in the toolbar.
• Accept the default settings and press OK.

The hierarchical clustering groups both samples (gel images) and expression profiles. The cluster hierarchy is shown in a tree display. As you can see, replicates are clustered together, indicating higher similarity, as we would expect.

Clustering Expression Profiles: Finding Correlated Proteins

Figure 5.53: Spots with similar expression profiles are clustered together. Support Tree clustering with Euclidean distance.

Discovering Patterns in Expression Profiles

Figure 5.54: Cutting a tree by a distance threshold. Use the slider to adjust the threshold.

• Right click and select Gene tree properties from the context menu.
• Use the slider to cut the tree at a certain distance from the root (fig. 5.54).
• Then check the Create Cluster Viewers checkbox and press OK.
• A new section called Gene Tree Cut is created in the left hand side of the display (fig. 5.55).

Finding differentially expressed proteins: Statistical Tests

Methods for statistical hypothesis testing in Delta2D are based on state-of-the-art algorithms that are applied in the context of DNA array analysis.

Figure 5.56: Result of applying t-tests (control vs. treated) to expression profiles. Profiles and images were clustered to better visualize differentially expressed proteins. P-values are based on 1000 permutations, false discovery rate is controlled to be 5 elements or less (with overall alpha=1%).

No normal distribution of spot intensities required

One has to keep in mind that the classical Student's t-Test makes the assumption that spot quantities within replicates follow a normal distribution which should be tested separately. Depending on the staining method you use and other factors, spot quantities within replicate gels may not be normally distributed. Therefore it is advisable to use one of the provided methods that are based on permutations.

In the t-Test options dialog, choose "p-values based on permutation" and either "Use all permutations" or "Randomly group samples" and enter "1000".

Controlling the False Discovery Rate

When applying statistical tests to 2-D gel data, one is faced with the so-called multiple hypothesis testing problem: For each expression profile, a separate test is done. Each test has a certain probability of giving a false positive result, i.e. a protein spot is declared to be differentially expressed while the difference was due to pure chance. The large number of tests can produce a high number of false positives. For example, in an experiment with 2000 spots per gel, an accepted false - positive rate alpha of 5% will result in 100 proteins that are found to be "differentially expressed" although the difference is the result of mere chance.

The MeV t-test module incorporated in Delta2D provides methods to control the proportion of false positives in the result set (False Discovery Rate - FDR). Overall, the False Discovery Rate approach allows one to strike a balance between the need to find statistically valid proteins of interest and the additional cost that is associated with following up on false positives.

In the t-Test options dialog, select "p-values based on permutations", "Stepdown Westfall and Young methods" and "maxT"'. Choose bounds for the number of false positive spots in the result set using the "number of false positive genes should not exceed". Alternatively choose a bound for the proportion of false positive spots in the result set, using the other radio button and text box.

Template Matching

With Template Matching, you can define a template for an expression profile and let Delta2D find spots whose expression profiles match the template. For example, in a time series experiment you might want to look for spots whose expression level increases with time.

a) b) Figure 5.57: a): Expression profiles matching the template. b): Comparison between template (blue line) and matching expression profiles.

Click on the PTM (Pavlidis Template Matching) button in the toolbar, or choose Analysis  Statistics  Pavlidis Template Matching from the menu. The Help button (labeled "i" on the bottom left of the dialog) gives more information about the options.

Principal Component Analysis (PCA): Grouping and Visualization

When you do Principal Component Analysis (PCA) on a set of gel images, you get a two- or three-dimensional visualization of the image set that is optimal in certain sense, i.e. it preserves the variation as much as possible. PCA works by taking spot intensities on every gel image and assembling them into a vector. So an experiment of 24 gel images with 1200 spots each would be represented as a cloud of 24 points in a space with 1200 dimensions. The goal of principal component analysis is then to find a projection of the point cloud in two or three-dimensional space such that as much as possible of the variation of the point cloud is preserved. One hopes that the gels from different samples will be in separate regions of the resulting diagram. The principal components can then be interpreted as "typical spot patterns" or "eigengels". Their coordinates can be analyzed in order to determine which spots are contributing most to the variance, making them candidates for protein identification and biological interpretation.

a) b) Figure 5.59: a): Principal component analysis of 24 gel images in 3 dimensions. Parallels have the same color. The view can be rotated by dragging with the mouse. Again, replicates are placed close together. b): The same principal component analysis of 24 gel images, projected onto the first two principal components. Treated and control samples (reddish vs greenish colors) can be separated.

Working with Sets of Spots

In the terminology of the TIGR Multiple Experiment Viewer (MeV), a cluster can be any set of expression profiles or samples (gel images). You can create new clusters by choosing Store Cluster in many displays of analysis results.

• In a clustering display, select the expression profiles of interest. In a hierachical clustering, you can select a whole branch of the dendrogram by clicking it in the tree. The corresponding expression profiles will be selected.
• Now right-click and select Store Cluster. The new cluster will be shown in the Cluster Manager under Gene Clusters.

Storing a sample cluster:

• In a hierarchical clustering, click on a part of the dendrogram for samples (column dendrogram), maybe you want to select a set of replicate gel images.
• Note how columns are selected in the heatmap display. Now right-click and select Store Cluster.
• A dialog opens that lets you define a name, comment and color of the cluster. You will have to select at least a color. Click the OK button.
• The new sample cluster should now be visible in the Cluster Manager. By default, the color of the cluster will now be shown on top of the heatmap column, and in other displays such as PCA (for samples).

In the Cluster Manager you can change any attribute, e.g. cluster colors, or whether the color should be used in displays. Note that clusters may overlap, but only one cluster's color will be used in displays.

Cluster A Cluster B

• Intersection: The new cluster contains only expression profiles that are present in each of the selected clusters.
• Union: The new cluster contains all expression profiles that were present in any of the selected clusters.
• XOR: The new cluster contains only expression profiles that are found exclusively in one of the selected clusters.

Statistical Analysis is Integrated with Image Analysis

When you select one or more spots in a heatmap display, the selection will be immediately visible in other parts of Delta2D, such as the Dual View, or the Gel Image Regions View. You can extend the selection to a range of rows by holding down the Shift key while clicking on the end of the range. You can add or remove a single row by holding down the Ctrl key while clicking on it.

If you have organized spots of interest in the Cluster Manager, you can use these directly in Delta2D. Just right click on a cluster and choose Select in Delta2D this will select the expression profiles in the cluster throughout all parts of Delta2D.

Getting a Spot Album of Relevant Spots

Using Delta2D's Spot Album Report, it is easy to show snapshots of the statistically significant spots you have found. All you have to do is mark these spots in the Delta2D project:

• Make sure you have selected the spots of interest.
• Switch to the Statistics tab of the Quantitation Table and choose Mark  Unmark all spots to unmark all spots that you might have marked previously.
• Then choose Mark  Mark selected spots.
• Then Reports / Spot Album. Note that the spot album may by quite large, as there is one image for each spot on each image. You can restrict the album to a single group by clicking on the "hide others" link in the group caption.

For more information about Reports see also section 3.6.

Overview of Statistical Methods

The following is a list of methods, for in-depth information please refer to the MeV manual and the original papers cited below.

Clustering

• Clustering can be applied to samples and / or expression profiles
• Hierarchical clustering and k-Means / k-Medians clustering
• Supports average linkage, complete linkage, and single linkage for determining cluster-to-cluster distances
• Supported distance metrics: Euclidean distance, Manhattan distance, Pearson correlation, Pearson uncentered correlation, Pearson squared correlation, Average dot product, Cosine correlation, Covariance, Spearman's rank correlation, Kendall's tau.
• Construction of support trees by resampling methods: bootstrapping (resampling with replacement), and jackknifing (resampling by leaving out one observation).

HCL - Hierarchical Clustering Eisen, M.B., P.T. Spellman, P.O. Brown, and D. Botstein. 1998. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95:14863-14868.

ST - Support trees (Bootstrapping) Graur, D., and W.-H. Li. 2000. Fundamentals of Molecular Evolution. Second Edition. Sinauer Associates, Sunderland, MA. pp 209-210.

KMC - K-Means Clustering Soukas, A., P. Cohen, N.D. Socci, and J.M. Friedman. 2000. Leptin-specific patterns of gene expression in white adipose tissue. Genes Dev. 14:963-980.

Template Matching

• Templates can be defined for expression profiles and samples.
• Templates can be defined interactively, from a given expression profile, or from a cluster.

PTM - Template matching Pavlidis, P., and W.S. Noble 2001. Analysis of strain and regional variation in gene expression in mouse brain. Genome Biology 2:research0042.1-0042.15.

Principal Component Analysis

• Principal component analysis is available for both samples and expression profiles.
• Three-dimensional and two-dimensional displays are available
• New clusters can be defined by dragging in a two-dimensional display.

Raychaudhuri, S., J. M. Stuart, & R. B. Altman 2000. Principal components analysis to summarize microarray experiments: application to sporulation time series. Pacific Symposium on Biocomputing 2000, Honolulu, Hawaii, 452-463. Available at http:/smi-web.stanford.edu/pubs/SMI_Abstracts/SMI-1999-0804.html

Statistical Hypothesis Testing

TTEST - T-Tests

• T-tests: one-sample, between samples, paired t-test
• Assuming equal or different group variances
• P-values can be computed based on normal distribution or using randomization.
• Corrections for multiple testing: Bonferroni, adjusted Bonferroni, Westfall-Young
• Control of false discovery rate
• Volcano Plot

Pan, W. (2002). A comparative review of statistical methods for discovering differentially expressed genes in replicated microarray experiments. Bioinformatics 18: 546-554.

Dudoit, S., Y.H. Yang, M.J. Callow, and T. Speed (2000).Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Technical report 2000 Statistics Department, University of California, Berkeley.

Welch B.L. (1947).The generalization of 'students' problem when several different population variances are involved. Biometrika 34: 28-35.

ANOVA - One-way Analysis of Variance

• P-values can be computed based on F-distribution or using randomization.
• Corrections for multiple testing: Bonferroni, adjusted Bonferroni, Westfall-Young
• Control of false discovery rate

Zar, J.H. 1999. Biostatistical Analysis. 4th ed. Prentice Hall, NJ.

TFA - Two-factor Analysis of Variance Keppel, G., and S. Zedeck.1989. Data Analysis for Research Designs. W. H. Freeman and Co., NY.

Manly, B.F.J. 1997. Randomization, Bootstrap and Monte Carlo Methods in Biology. 2nd ed. Chapman and Hall / CRC , FL.

Zar, J.H. 1999. Biostatistical Analysis. 4th ed. Prentice Hall, NJ.

References

Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J. TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003 Feb;34(2):374–8.

Alter O, Brown PO, Botstein D (2000) Singular value decomposition for genome-wide expression data processing and modeling. Proc Natl Acad Sci U S A 97:10101–10106

Holter NS, Mitra M, Maritan A, Cieplak M, Banavar JR, Fedoroff NV (2000) Fundamental patterns underlying gene expression profiles: simplicity from complexity. Proc Natl Acad Sci U S A 97:8409–8414

TIGR Multiple Experiment Viewer (MeV): http:/www.tm4.org/mev.html

TIGR MeV manual: http:/www.decodon.com/Support/Documentation/MeV

5.12 Project Matrix

In the Project Matrix, previously known as 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, respectively.

Figure 5.61: Details in project table

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 Image Properties . . . Shows properties of the gel and add a comment. Fuse all Images Create a new image, by fusing all images of your project (see section 7). 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 3.4). Spot Color Coding Use the selected gel image as basis 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 the group. Table 5.6: The context menu in the project table header

5.13 Arrange Windows

Delta2D is based on a modern window manager that allows for easy reconfiguration of the window setting.

You can drag the windows to other positions in the main Delta2D window or you can undock them so that you can freely arrange them on your desktop.

To drag a window click on its title bar and move it around. If you place the window to an alternative valid position the new position is highlighted with a frame. Drag the window and it will keep its new position until you change it again. Closing and re-opening does not affect the position.

To undock a window right-click on its title bar and choose Undock Window to seperate it from the Delta2D window.