Seg2Dgel Program Frequently asked questions (FAQ)

The following are some frequently asked questions. This is a list of some of the questions that we will be answering. Please send us your questions about Seg2Dgel so we can add them and the answers to this list.

FAQ

How do I specify the gel to segment? You can specify the gel to segment using the (File | Open gel file) menu command or using the Browse file button in the Options window that you bring up using the Edit options button in the Report window.

What is a project and a project directory? What goes into this directory and can I have more than one? The segmenter (and other Open2Dprot programs) work in the context of a project. A project is a set of data to be analyzed together - typically a set of gel experiments. The projectcontains several subdirecties (ppx/ - containing the original images, cal/ - containing optional calibration files for each original gel, tmp/ - containing any output images generated by the segmenter, and xml/ - containing the spot list gel segmentation file and other text files).

What is an experiment? We define an experiment as a set of gels that may include different conditions

What is a command line switch? The Open2Dprot programs, of which Seg2Dgel is one, are designed to be run either under a batch mode or a graphical user interface window mode. Command line switches are the common control mechanism. We have adopted the Unix convention that switches start with a "-" and may have additional parameters. For example, -thrArea:10,2000 specifies that only spots with a minimum area of 10 pixels through 2000 pixels should be accepted. See the list of command line switches for details.
How are switches used? See the command line examples.
How do I change the command line switch options? Bring up the Options window using the Edit options button in the Report window.
How do I adjust the spot sizing parameters (area, density, OD range)? Bring up the Options window using the Edit options button in the Report window. Adjust the threshold sliders to the parameters you desire. Then press the Set new default button. This returns control back to the Report window. Then press the Segment button to resegment the image. After it finishes, use the Image viewer to see how well it segmented the gel using the new parameters. You can repeat this process to titrate various parameters to arrive a a "good" set of parameters to use.
How do I handle requiring different sizing parameters for different gels? If the gels are similar, then you may be able to titrate an examplar gel and use it on the rest of the gels. Otherwise, you might have to "tweek" the parameters for some of the outlier gels.
Can I save the options that I have just changed and apply them to other gels? Yes. When you exit the segmenter, it saves the new parameters in a {project directory}/Seg2Dgel.properties file. [Status: disabled while in pre-alpha testing level.]
What is an OD calibration and where do I get it? A sample image calibration defines the mapping of the image pixel grayvalues in terms of another quantity such as optical density, counts/minute, etc. The calibration is found in the accession database entry for the sample. The Open2Dprot sample accessioning program Accession will contain the sample calibration procedure.
How do I see the image results of a segmentation I am done segmenting? Press the Image viewer button in the Report window or use the (View | Image viewer menu command.
What is a Sample Segmentation File (SSF). It is a disk file that contains the numeric data for the list of spots just segmented. The format depends on the -ssfFormat:{F | G | T | X} command line switch.
How do I see the list of spots numeric data results of a segmentation I am done segmenting? Use the (View | Show SSF report menu command to display the data in the Report window. Alternatively, you can use the (View | Show SSF browser menu command to display the data in another browser (depends on how you computer displays .txt and .xml files).
How do I see use the Image Viewer to look at the numeric spot feature data for a particular spot? Bring up the Image viewer and click on a spot. The data appears at top of the Image viewer. Hint: set the subwindow size to 1x1 as the window data is distracting. If you set the Use drag checkbox, then as you drag the mouse over the image, it will update the spot data. If you set the Log to Report checkbox, then as you click on spots, it will copy that data to the Report window for a more permanent record.
How do I see use the Image Viewer to look at a subwindow of numeric pixel data both in decimal and in optical density (OD if calibration)? Bring up the Image viewer. Then set the subwindow size using a pull-down chooser at the bottom of the window (1x1, 3x3, ..., 21x21) pixels. Then click on the spot you are interested in the image. To view the data as decimal, set the "Decimal radix" choice. If the image is calibrated in optical density, you can view the OD values by setting the radix to "OD radix". If you set the Use drag checkbox, then as you drag the mouse over the image, it will update the window data. If you set the Log to Report checkbox, then as you click on spots, it will copy that data to the Report window for a more permanent record.
When segmenting a sample, how do I stop the segmenter when it seems to have gotten stuck or is taking too long? Press the Stop segmentation button in the Report window.
How do I generate an XML output file? Set the command option to the -ssfFormat:X using the Options window that you bring up using the Edit options button in the Report window.

Contact us Seg2Dgel is a contributed program available at open2dprot.sourceforge.net/Seg2Dgel Revised: 10/04/2005