Frequently Asked Questions

A. BioDiscovery does not support the use of proprietary file formats. Rather, BioDiscovery has adopted the use of tab delimited text files. Microsoft Excel files can easily be saved as tab delimited text files as outlined below.

To save an Excel Doc as Tab Delimited Text:

1. With the Excel document open, choose Save as... from the File Menu.
2. Select Text (Tab Delimited) from the Save as Type Option at the bottom of the Save as Window.
3. Specify a File name and location.
4. Click Save.
5. Microsoft will warn that certain elements cannot be saved in this format. Simply accept any warnings to complete the process.
For additional information on the required text format, please see the documentation accompanying the software.

A. All BioDiscovery products are written in Java. Java can only see drive letters, not network locations. Therefore, the simplest solution is to map a network location to a drive letter.

Under Windows, you can browse the network, right-click on the folder that contains the files and select Map Network Drive. Map it to a letter that is not being used by anything else (like "N" for Network or whatever you want). Then, when the BioDiscovery software asks for a file, simply select the N: drive and it will load the files across the network without transferring them locally first.

A. BioDiscovery currently offers two ways to register newly purchased software:

1. via Fax - All new purchases, with the exception of additional floating licenses, will contain a Product Registration Form. Fill out this form completely and fax to BioDiscovery Corporate Headquarters.
2. via Online Form - Process to the online registration form and complete all required field. Be certain to check the YES box to agree to the Software License Agreement. Register at:

Please address any additional questions or registration problems to support@biodiscovery.com.

A. The permissions are set up correctly by our software when you install it, but only if you are using an account that has administrative privileges.

You can always un-install and re-install using an account that has administrative privileges.

A. There are a few potential causes of this problem. When the little screen appears then stays visible for a minute then disappears yet the software does not start, this is an indication that the license cannot be checked out and as a result, the software will not start. There could be several reasons for this: 1.) The flexlm license server is not running. In this case, go to the location where flexlm is installed and run lmtools.exe. Within this software is the ability to start and stop the license server. Be certain the server is started. 2.) Turn off any firewall software running on the computer as this may block network ports used by the licensing software. If this still does not solve the problem: 1.) Run lmtools.exe. Click on the Server Status tab. Click on the perform status inquiry button. This should tell you if there is anything wrong with the license server. 2.) Provide the GeneSight.log file, located in the GeneSight Directory, to BioDiscovery support so that we can determine if there is another problem.

A. It sounds like your GeneSight.ini, ImaGene.ini, or CloneTracker.ini has been set to "read-only" (saving the ini file as an attachment from Outlook will cause this problem). In this case, your software will generate a new "Entry Code" every time you run the license manager since it cannot save the code to the ini file.

In order to make sure your ini file is not "ready-only", you need to open your product directory ( C:\GeneSight for example), find the ini file, right-click on it, select "Properties" on the pop-up menu and confim that the "Read-Only" attribute is NOT checked. If you cannot see the ini file in your product directory, then you will need to show "System Files" by clicking on Tools, Folder Options, View tab, and make sure "Hide protected operating system files" is NOT checked.

A. BioDiscovery software is dependent upon Sun Microsystems Java Runtime Environment version 1.3 or higher. As a result of this, BioDiscovery software installs and modifies several files within the Java JRE home directory. There are times when due to configuration errors or changes to the host computer configuration, these files become either misplaced or corrupted. An alternative cause can also be the installation of a different JRE on the same computer. In this circumstance, ImaGene cannot load integral system files resulting in the Kernel Error Message.

Possible solutions include:

1. After backing up the software license, uninstall then reinstall the software. During the reinstallation, BioDiscovery software can then adapt to any new changes to the system configuration. If possible, the Java Runtime Environment (v1.3 or higher) should also be uninstalled and reinstalled prior to reinstalling any BioDiscovery software.

License File Names
• ImaGene - imagene.ini
• GeneSight - genesight.ini
• CloneTracker - CTlicense.ini

2. Check the system path and/or Java Home environment variables to be certain these point to the correct JRE 1.3 directory. Use caution when changing these values as they may affect other software on the computer as well. If uncertain, please contact your network administrator or contact BioDiscovery support.

A. No. Currently, all image analysis software use unique, proprietary formats for saving data.

A. Local Flexibility defines the radius, measured in pixels, that ImaGene is allowed to search for spots. The origin for the search is the initial spot location as determined by grid placement. Usually, a value of 3 - 5 pixels is the typical setting. Global Flexibility is an indication of the extent to which ImaGene should deform the grid to match a given set of spots. Most users should set sliding bar to the middle.

A. Select the magnification tool and double right-click.

A. ImaGene has been designed from the beginning for muti-channel analysis. In fact, the number of images that can be processed is limited mainly by the amount of memory present within the computer.

A. There is no fixed limit. You should have at least a 10 levels on a system that meets our minimum requirements, but the actual amount will vary depending on the RAM of your system.

A. Each input file must have a seperate name, otherwise the output file will be written twice (the 2nd image data overwriting the first).
For example, C:\Test1\MyData.tif and C:\Test2\MyData.tif would result in only a single "MyData.txt" file (with only the C:\Test2\MyData.tif results).

A. The .sst file contains links to the images that generated it. In ImaGene 4.2, these links are the absolute paths to the images. This means that the files MUST be in the same location as they were originally.

For example, if the files were stored on the original computer as:

• C:\ImaGene\Files\SampleImage1.tif
• C:\ImaGene\Files\SampleImage2.tif
• C:\ImaGene\Files\Sample_Data\SampleA.txt
• C:\ImaGene\Files\Sample_Data\SampleB.txt
• C:\ImaGene\Files\Sample_Data\Sample.sst

• D:\ImaGene\Files\SampleImage1.tif
• D:\ImaGene\Files\SampleImage2.tif
• D:\ImaGene\Files\Sample_Data\SampleA.txt
• D:\ImaGene\Files\Sample_Data\SampleB.txt
• D:\ImaGene\Files\Sample_Data\Sample.sst

• C:\ImaGene\Files\SampleImage1.tif
• C:\ImaGene\Files\SampleImage2.tif
• C:\ImaGene\Files\Sample_Data\SampleA.txt
• C:\ImaGene\Files\Sample_Data\SampleB.txt
• C:\ImaGene\Files\Sample_Data\Sample.sst

A. Image processing in ImaGene is very straightforward and normally only takes a few steps. If the images are in good quality, the steps are: Loading images, Creating and placing grid, loading Gene ID file, Clicking the Automatically place grid button, Clicking the Auto Adjust Spots button, Quantification, Saving results.

Of course, user has to spend some time to set the right configuration parameters in ImaGene Settings before doing image processing. Sometimes when the images are not in good quality, user also has to manually adjust the grids or spots for portions of the images.

A. The min. and max. diameters of image spots affect the grid placement and spot finding, and in turn affect the segmentation. For this reason, it is important to get a relatively accurate measurement before creating a grid. It is better to zoom up a group of smallest or largest spots, measure them using the ruler tool, and take the average. Usually, within 2 to 3 pixels in accuracy is good enough.

A. There is no fixed number here. Normally, one click of the Auto Adjust Spots icon is enough. But you can do it a few times if you do not like the results.

A. This phenomenon puzzles users and makes them redo spot finding again and again. In fact, the grid circle only shows where the image spot is and it does not have to cover the entire spot. After quantification, go to the segmentation tab for each image (image name seg tab) to check the segmentation of each spot. As long as the red segmentation line covers each spot, the spot finding works.

A. Each value corresponds to a different type of flag. These flags are represented visually on your quantified data display by different markers. 0 = No Flag (no marker) 1 = Manual Flag, No Type (red X) 2 = Auto-Flag, Empty Spot (green X) 3 = Auto-Flag, Poor Spot (green +) 4 = Auto-Flag, Negative Spot (green -) 5 = Manual Flag, Empty Spot (red X) 6 = Manual Flag, Poor Spot (red +) 7 = Manual Flag, Negative Spot (red -)

A. The wrangle feature of ImaGene applies new, stricter constraints to the results of spot localization without requiring further spot finding. Essentially, this allows users to reduce the spot search radius without redoing the spot finding. The benefit of this feature is to assist processing for those with either slower computer hardware or for those with numerous spots.

A sample application would be to perform spot finding for a grid geometry on an array image with a local flexibility set a large number of pixels. After spot finding, if the resulting circle placement has high variability, the Local Flexibly can be reduced. After reducing Local Flexibility, click the Wrangle Button to apply the new setting without waiting for spot finding to be performed again.

Most users with recent computer hardware need not employ this feature as it has become largely unnecessary.

A. ImaGene supports:

Tiff - (*.tif)
Bas - (*.inf, *.bas, *.img)
Gel - (*.gel)

Currently, there are no plans to add additional formats.

A. ImaGene supports:

Tiff - (*.tif)
Bas - (*.inf, *.bas, *.img)
Gel - (*.gel)

Currently, there are no plans to add additional formats.

A.

• Negative Spot = signal is lower than ambient background (usually a bad thing)
• Empty Spot = signal is equal to ambient background (or very close)
• Good Spot = singal is higher than ambient background

A. ImaGene Premium comes with an optional Automation Module that allows the quantificiation of multiple images in a batch. This allows time-consuming quantification to be performed automatically during time when the computer is not being used by researchers.

A. The SST file produced by ImaGene is a proprietary, binary file containing information about the processing that has occurred.

The SST file is used by ImaGene to review the VISUAL results of previous image processing. The SST file contains information about segmentation, quality measures, grid placement and other data generated as a by product of processing. The SST file allows ImaGene to reload and consequently display VISUAL indicators of previous processing.

Since the SST file is only necessary to review the visual results of processing, users who are only interested in the quantified values (mean, median, etc) can simply delete this file. ImaGene always generates standard text files with the quantified values for each image processed. This means that the text data files are saved and used for later analysis in such products as GeneSight.

The SST file is not designed to be loaded into GeneSight or any other data analysis software packages. The information contained within the SST file is of no value to other programs.

The SST file can often grow quite large in size, often times becoming larger than the original image. This is common and is due to the amount of visual information and quality measures contained within.

The SST file aids Flex Pack and Automation module users the most as it allows for easy review of Batch Processed Images. For additional information on the SST file, please consult your ImaGene documentation.

A. A template is composed of both a grid and a GeneID file. A grid is only the geometry of the array without any clone information.

Both templates and grid files can be created from the Grid Menu along the menu bar.

Both items can be used when batch processing.

A. A template is composed of both a grid and a GeneID file. A grid is only the geometry of the array without any clone information.

Both templates and grid files can be created from the Grid Menu along the menu bar.

Both items can be used when batch processing.

A. ImaGene-Lite has exactly the same features and functions as ImaGene with these exceptions: 1. No grid creation, saving and user-definition functions are provided with ImaGene-Lite, only one or more template files (which include the exact grid and gene identifiers) for specified brands and types of commercially-available filter, membrane, or slide arrays. 2. The GeneSight-Lite module and its functions(inter-channel or inter- experiment ratioing, plotting, and comparison) are not included in ImaGene-Lite.

A. ImaGene Premium includes better Auto-Segementation and additional quantification measurements:

• Shape Regularity - how uniform a spot is
• Ignored Area - number of pixels discarded by segmentation
• Ignored Median - median value of pixels discarded by segmentation
• Spot Area - size of spot before segmentation
• Perimeter-to-Area ratio - how circular a spot is
• Position Offset - distance of actual spot from ideal location on grid

A. These are quanitification flag markers:

• X = empty spot
• + = poor spot
• - = negative spot
• Red = manually selected
• Green = selected by parameters

A. Spot circles are colored using gradual scale from blue to red, with blue indicating no contamination and red indicating that contamination is present.
If both signal and background contamination confidence tests are chosen, the colors represent the equally weighted sum of two confidence values. If one test is selected, then only that result will define the coloring.

A. ImaGene currently supports the following templates:

Agilent Technologies
Corning
CloneTech
Research Genetics
NEN Life Sciences
Incyte Genomics

New templates may be added. As a result, please contact support for a listing of current templates.

A. ImaGene currently supports the following templates:

Agilent Technologies
Corning
CloneTech
Research Genetics
NEN Life Sciences
Incyte Genomics

New templates may be added. As a result, please contact support for a listing of current templates.

A. The Preview Window allows users to see the results of segmentation prior to quantifying an image or images. The purpose of this window is to be certain that the proper settings have been established so that ImaGene removes contamination, such as dust, properly.

ImaGene uses patent-pending statistical methods to determine whether individual pixels belong to the signal or background regions. Green represents pixels that will be used in calculating a background value. Red represent pixels that will be used in calculating a signal value. Pixels that are neither red nor green are ignored and thus not used in the calculation of mean, median, etc values.

Ultimately, the Preview Window helps user establish optimal parameters ensuring high quality, consistent data.

A. When ImaGene opens images for analysis, it reads information containded within the image file that explains which pixels represent high and low intensities. Users often assume that the visually white pixels always represent high values; however, this may or may not be the case depending on the information provided within the image file.

The problem is that there are times when the image generation software does NOT include information describing what values white and black pixels should represent. The result is that ImaGene displays this information "backwards", meaning what the user expects to contain high values in reality is being displayed with the inverse value.

ImaGene provides a handy solution to this problem. On the Main User Interface, located under the Image Tab, is an Invert Checkbox. Select this checkbox to invert the value of pixels within the image. Visually, the image will "flip" causing high and low intensity pixels to change in the image window.

Note: Selecting the Invert Checkbox truly inverts the values of the images which is reflected in the quantified data files. To "flip" the view only for visual puposes, not affecting the quantified values, only select the Reverse Display Image Checkbox located at the bottom of the Image Tab.

A. You must have the file "license.dat" in the same directory as the ImaGene executable. Also, be certain the file netlicense.txt contains the correct path to the imagene.ini license file.

A. The ability to run batches is controlled by the BatchUsers.txt file. This file contains all the names of users who are eligible to run ImaGene in batch mode on that particular computer.

The location of this file is within the data\imagene directory inside Java Home. Typically, this location is:
C:\Program Files\JavaSoft\JRE\1.3\data\imagene
although the exact location depends on installation selections.

The usernames contained within this file must be the usernames used to login to the computer. For example, if John Smith logs into his computer as jsmith, then BatchUsers.txt must contain the entry jsmith. If multiple users log into this computer and they wish to run ImaGene in batch mode, then either all usernames must be entered or the use of the keyword anyone is also permitted. The keyword anyone permits all users to operate ImaGene in batch mode.

In environments where numerous users exist and/or security is important, Adminstrators are encouraged to set file privledges on BatchUsers.txt to limit its modification.

A. The ability to run batches is controlled by the BatchUsers.txt file. This file contains all the names of users who are eligible to run ImaGene in batch mode on that particular computer.

The location of this file is within the data\imagene directory inside Java Home. Typically, this location is:
C:\Program Files\JavaSoft\JRE\1.3\data\imagene
although the exact location depends on installation selections.

The usernames contained within this file must be the usernames used to login to the computer. For example, if John Smith logs into his computer as jsmith, then BatchUsers.txt must contain the entry jsmith. If multiple users log into this computer and they wish to run ImaGene in batch mode, then either all usernames must be entered or the use of the keyword anyone is also permitted. The keyword anyone permits all users to operate ImaGene in batch mode.

In environments where numerous users exist and/or security is important, Adminstrators are encouraged to set file privledges on BatchUsers.txt to limit its modification.

A. Typicaly, when ImaGene stops processing in the middle of analysis, there is an insufficient amount of memory to continue. The simplest rememdy is to the open the ImaGene.properties file located within the ImaGene\Jexpress folder and increase the -mxXXXm value. This value can be set up to aproximately -mx1024m (1024 MB). Please note that while this change may allow analysis, without enough matching physical RAM in the computer performance will be geatly dimished.

A. The segmentation tab, displaying the segmentation results, is only available within Premium Pack or Automation Module versions of ImaGene. This feature is enabled within demo mode as well. If you you feel you have purchased these modules and the segmentation tab is still not visible, please contact BioDiscovery support for additional information.

A. The segmentation tab, displaying the segmentation results, is only available within Premium Pack or Automation Module versions of ImaGene. This feature is enabled within demo mode as well. If you you feel you have purchased these modules and the segmentation tab is still not visible, please contact BioDiscovery support for additional information.

A. Perform the following steps below to troubleshoot this problem:

License Manager Server:

1. Determine if the following processes are running. On Windows, one should see biod.exe and lmgrd.exe are running. If either process is not running, continue to Step #2.

2. Bring up the License Manager console. On Windows, start lmtools.exe. Verify the settings are correct. Refer to FAQ #586 for more information.

3. Restart the server, and return to Step #1 above.

License Wizard:

1. Open the License Wizard. Make sure all the information is entered correctly. Refer to FAQ #587 for more information.

Check for Demo License:

1. Check for a demo license file, “local.lic under the main BioDiscovery product installation. If one exists, remove it.

A. No. Any change made to the license file, renders it unusable.

A. Yes, one can download the Linux and Windows versions of the License Manager from the web: http://www.biodiscovery.com/downloads.asp.

A. Yes, the License Manager can run on Linux as well.

A. No License Manager is needed to run the products using a Demo license. The License Manager is only used for normal licenses.

A. Yes, the demo license must be removed in order for the BioDiscovery products to use the normal license. If not removed, the product will default to the Demo license.

A. The BioDiscovery products run in one of two modes: NodeLocked or Floating. The necessary steps to configure the BioDiscovery products are as follows:

1. If a Demo license exists, delete it.

2. Open License Wizard (Start -> Programs -> BioDiscovery’s software (ImaGene or GeneSight) -> License Wizard).

3. For NodeLocked installations, enter the User Name, Institution Name, and Serial Number and select “SaveNodeLocked.



4. For Floating installations, Enter the User Name, Institution Name, Serial Number, and Server Name. In the example below, the server name is “BIOSERVER. Lastly, select “Save Floating.

A. After the License Manager is installed, the steps to configure it are as follows:

1. Save the license file, “license.lic, in the appropriate License Manager directory. The default on Windows is C:\FLEXlm.

2. Start the FLEXlm console. On Windows, start lmtools.exe, located under the License Manager’s main directory.



3. Configure the settings as above on the Config Services tab. The example above reflects all default values.

4. Click Start Sever in Start/Stop/Reread tab. If the license server was started before, Stop Server and Start Server again.

A. You can visit http://www.macrovision.com/solutions/esd/support/enduser/TOC.htm for a complete description of the FLEXlm license manager.

A. The demo license file “local.lic, usually is sent as an email attachment. After the BioDiscovery product installation process is finished, one must put this file under the main product directory; the default for Windows, for instance, are C:\ImaGene or C:\GeneSight.
NOTE: the product looks for the specific file “local.lic, so one must be careful when using certain email systems that attach other symbols or numbers to file names (e.g., local(1).lic).

A. A normal license must reside on the computer that is running the License Manager (FLEXlm). If installed as NodeLocked, it will reside on the same machine as the product itself. Otherwise, it must be stored on a machine running the License Server, i.e. Floating mode. Refer to FAQ #473 for more details.

A. The FLEXlm License Manager Server can be installed one of two ways:
1. As part of the BioDiscovery software installation
2. It can also be downloaded from the BioDiscovery website and installed separately

A. This requires a new BioDiscovery product license file to be generated. Contact support@BioDiscovery.com to generate the new license file. Refer to FAQ #583 for more information concerning License Manager installation.

A. When running in demo mode, make sure the demo license file, local.lic, is saved in the main product directory (refer to FAQ #471 for more information). Otherwise, check the appropriate License Manager directory (refer to FAQ #473) for more information. Lastly, make sure the license file has not expired.

A. The “local.lic Demo license is not installed under the appropriate product directory. Refer to FAQ #471 for more information.

A. The demo license does not require a License Manager. Therefore, it should be stored in the appropriate BioDiscovery product directory, for example, the Windows default are C:\ImaGene or C:\GeneSight. A normal product license, however, needs to be stored under the root License Manager directory, i.e. the default for Windows C:\ FLEXlm.
NOTE: the License Manager only looks for the “license.lic filename. Again, one should make sure the file name is not tampered with when copying from an email application.

A. When the License Manager runs in NodeLocked mode, the License Manager Server is running on the same computer as the BioDiscovery product. When running in Floating mode, the License Manager Server runs on a different machine.

A. “local.lic is for Demo purposes only and “license.lic is a normal license file. A Demo license is stored under an installed BioDiscovery product, while a normal license is stored under the appropriate License Manager directory.

A. The License Manager needs to be installed and configured before one can use a normal product license. Refer to FAQ #583 for more information.

A. There are two configurations:

1. Nexus Copy Number Standard Edition – A streamlined version that might be more applicable for clinical and cytogenetic laboratories.
2. Nexus Copy Number Professional Edition – Includes additional features enabling rapid research:
   1. External data Integration
   2. Statistical Class Comparisons
   3. Enrichment Analysis
   4. Unsupervised Clustering

A. Yes. Here are examples of some popular commercial BAC-arrays supported by Nexus Copy Number: Empire Genomics, Perkin Elmer, BlueGnome.

A. Yes. Here are examples of some popular commercial oligo-arrays supported by Nexus Copy Number: Affymetrix, Agilent, Illumina, Roche NimbleGen,

A. Absolutely. Data from any number of different platforms can be integrated, visualized, and analyzed together in Nexus Copy Number.

A. Yes. Nexus Copy Number Professional Edition allows you to incorporate external data such as miRNA and gene expression to identify genomic hot spots.

A. Yes. Nexus Copy Number Professional Edition allows you to incorporate external methylation data to identify genomic hot spots.

A. Yes. Nexus Copy Number allows you to add your own algorithms written in R or bring data that has already been segmented for further processing.

A. Yes. Nexus can quickly identify regions of genomic change that can distinguish between two populations (e.g. affected and unaffected individuals) and provide statistical confidence measure for each region.

A. The highest density arrays that are available on the market can be used. Nexus Copy Number can efficiently process the Roche NimbleGen HD2 arrays with over 2.1 million probes or the Affymetrix SNP 6.0 array with 1.8 million probes.

A. You simply select files you want to load using the file chooser and then click on the “Load Data” button to load the data. In rare cases such as when loading dye-swap data or replicate data, you will create a Sample Descriptor (a tab de-limited text file) which will specify the sample types and files to load.

A. An unlimited number of samples can be analyzed together. Some of our customers have used several thousand very high-density arrays in a single project on a typical desktop computer.

A. Nexus Copy Number is a desktop software program for analysis of DNA copy number variation from CGH and SNP microarray data. In a user-friendly and efficient fashion, it allows users to detect the segments of the DNA that have been lost or amplified.

A. Nexus Copy Number supports all array platforms, such as Affymetrix, Agilent, Illumina, Roche NimbleGen, Empire Genomics, custom BAC arrays and more. It also supports data generated by various array image analysis software tools such as GenePix, ImaGene, and BlueFuse.

A. Platforms Supported: Windows Win2k/WinXP, OS X, Linux
Minimum: 1.0 GHz Pentium or G4, 512 MB RAM
Recommended: 2.0 GHz or faster Core 2 Duo, 2GB or more RAM

A. Nexus Copy Number offers the following algorithms for making calls.

• Rank Segmentation: A robust variation of the well-known Circular Binary Segmentation (CBS) algorithm where the probe ranks are used to minimize the effect of outliers and drastically improve performance.
• SNPRank Segmentation: An extension of the Rank Segmentation algorithm where B-Allele Frequency values are also included in the segmentation process generating both copy number and allelic event calls.
• FASST Segmentation: A novel Hidden Markov Model (HMM) based approach that unlike other HMM methods does not aim to estimate the copy number state at each probe but uses many states to cover possibilities, such as mosaic events, and then make calls based on a second level threshold
• SNP-FASST Segmentation: An extension of the FASST algorithm but adding many more states to cover events related to the B-Allele Frequency values to make copy number and allelic event calls.