1. What does GeneMarker do?

   GeneMarker is a software program designed for DNA fragment analysis with gel and capillary electrophoresis traces, such as AFLP, Genotyping, STR (Short Tandem Repeat), Snap-Shot, and MLPA analysis. It is also used for quantification of DNA fragments.

2. Which instrument data does the software support?

   GeneMarker software supports most of the data formats from the electrophoresis instruments. The software reads the RSD, ESD and SCF formats used in MegaBACE capillary systems from Amersham Biosciences or GE Healthcare; FSA, AB1 and ABI formats used in slab gel or capillary systems from Applied Biosystems; SCF format used in BaseStation Analyzer from MJ Research Inc.; TIF format used in FMBIO imaging systems; SMR and SMD formats in SCE capillary systems from SpectruMedix.

3. What are the advantages of GeneMarker?

   The main advantages of the software are ease of use and accurate, quick sample analysis. Automated features of the software include correction of saturated peaks and instrument spikes, removal of pull-ups caused by matrix residue, and baseline subtraction to eliminate noise. The allele calls are done with the pattern recognition algorithms so that false positives are reduced. The majority of functions have been completely automated, using a "smart" software approach to eliminate the need for constant analyst intervention, and to improve analysis to analysis consistency.

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2. Software Parameter and Preference Options Questions

1. How do I set the parameters for different types of analysis?

   Click the icon to begin the Run Wizard and a window titled "Template Selection" will appear. Under "Analysis Type" choose Fragment (default), SnapShot, or AFLP. To select the "Size Standard" and "Panel" for fragment analysis please see 3a and 3d-3e.

2. How do I set the analysis parameters for "Standard Color"?

   Click the icon to begin the Run Wizard and a window titled "Template Selection" will appear. The default "Standard Color" is red, but if your data uses another color, you may chose the appropriate color (blue, green, yellow, red, orange) from the Standard Color drop-down box.

3. How do I choose the correct "Size Standard?"

   In the Run Wizard, please select one size standard that you are using, such as Gene Scan 500, High Definition 400, Internal Lane Standard 600 etc. The software will use this size standard to align all of the lanes. If you don't know which size standard is used in your experiments, please consult the standard manufacturer or set up your own standard. You may create your own standard by loading the raw data and going to Tools>> Size Template Editor. Choose the size standard that best matches your samples from the Size Standards tree located at the left of the screen. You may also import your own size standard or create a new size standard under the File menu. The first time you run the data, click the "Run" icon and select your chosen size standard i.e

4. How do I choose the correct "Panel?"

   For first time users, choose None and run the analysis to perform ‘Size Calling.' After ‘Size Calling' is completed, you can either create a new panel (see 3e) or import a panel under File >> Import Panels. To modify an existing panel please see 3d.

5. How should I set the "Peak Detection Threshold?"

   Click the icon to begin the Run Wizard and go to the second window labeled "Data Process-Fragment Analysis." There are three parameters for peak detection:

   1. Intensity: Minimum peak height. The recommended setting is 100 (For AFLP analysis it is recommended to lower the threshold).

   2. Percentage: Minimum percent of allele peaks to the highest peak in the lane. The recommended setting is between 1 and 5.

   3. Local Region Percent: Defines the peak detection threshold based upon the percentage of the highest peak in that locus. If the threshold is set to 33% for example, the height of all allele peaks must reach at least 33% of the height of the highest peak in that particular locus. For Fragment and Snapshot analysis, the recommended setting is 30%. For AFLP analysis, the recommended setting is 1%.

6. How should I set the Stutter Peak Filter (%) threshold?

   Click the icon to begin the Run Wizard and go to the second window labeled "Data Process-Fragment Analysis." The Stutter Peak Filter allows the removal of forward and reverse stutter peaks commonly caused by the chemical reactions. The settings are in % of the primary peak. The default settings will remove stutter peaks that are 90% forward of the primary peak, and 30% following the primary peak (recommended setting).

7. How should I set the score parameters?

   Click the icon to begin the Run Wizard and go to the third window labeled "Additional Settings-Fragment Analysis." We recommend setting the parameters as follows: the software will reject allelic peaks with a score below 1, check those with a score between 1 and 10, and pass the allele peaks with a score above 10, however, these settings may be adjusted for your preference. Once the analysis has run, the allele report, located on the far right of the screen, will show all of the detected alleles with a particular symbol. Depending on the previously set parameters, allele peaks with a "" symbol have very low confidence, those with a "" symbol fall within the "check" range and should be verified, and those represented by the "" symbol have high confidence and fall within the "pass" range.

   The user can also set the level of confidence for preceding peaks at the right side prior to accepting a peak for an AFLP application. We recommend setting the score to 10. This requires that two peaks to the right of the examined peak must have a score of at least 10 for the peak to be accepted.

8. How do I view multiple electropherograms at once?

   Go to View >> Preference and click the Display Settings Tab. The View Settings tab is used to set the precision of the data, in tenths of base pairs, the maximum number of open charts (set to 2), the maximum number of lane electropherograms that can be viewed in one screen, and finally the maximum numbers of Allele label layers.

   Range of Options:
   Peak Size Labeling Sensitivity: 0-2 decimal places
   Chart Setting Ranges:
   Max Number of Open Charts: 2
   Charts per Screen: 1-8
   Allele Labeling Layers: 1-6

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3. Template Questions

1. How do I pick the correct size standard?

   In the Run Wizard, please select one size standard that you are using, such as Gene Scan 500, High Definition 400, Internal Lane Standard 600 etc. The software will use this size standard to align all of the lanes. If you don't know which size standard is used in your experiments, please consult the standard manufacturer or set up your own standard. You may create your own standard by loading the raw data and going to Tools>> Size Template Editor. Choose the size standard that best matches your samples from the Size Standards tree located at the left of the screen. You may also import your own size standard or create a new size standard under the File menu. The first time you run the data, click the "Run" icon and select your chosen size standard i.e

2. How do I create my own size standard in the Size Template Editor?

   To create a new Size Standard, go to Tools>>Size Template Editor. Then either click the button or click File >> New Size Standard in the toolbar. Name the new Size Standard and then insert size standard peaks where desired by right clicking on that location in the upper panel. The lower panel represents the different samples and you may choose the sample to be displayed by double clicking on the sample name located in the Sample File Tree. A checkmark indicates the sample is displayed in the panel and a blank page indicates it is not displayed:

3. What is "Size Calling" and how do I complete it?

   ‘Size Calling' is a function which compares the sample files against a selected size standard. ‘Size Calling' is not necessary to run the Size Template Editor The Size Template Editor can be run before or after the ‘Size Calling' is performed, depending on your needs. If your data requires a completely customized size standard, you should not run ‘Size Calling' first. In this case, the software allows you to create an entirely original size standard for future use through the Size Template Editor. If your project requires a modification to a typical size standard, you can run 'Size Calling' with the size standard you wish to modify, then use the Size Template Editor to make the necessary changes.

   The ‘Size Calling' function is activated by clicking the green play button in the GeneMarker toolbar. This launches the Run Wizard . The first page is for Template Selection. You must be sure to use the correct size standard for the data and click "Next." The next page is to specify the data process options that you desire, and then click "Next." The last page is for additional setting, then click "OK.

4. How do I use the Panel Editor?

   Prior to using the Panel Editor function, you need to have already started the GeneMarker application and either opened an existing project or opened specific stored data. ‘Size Calling' must be performed on the data before viewing the data in Panel Manager. Once this is complete, Panel Editor's complete functionality is available to you. Panel Editor can be accessed by selecting the Tools dropdown from the main GeneMarker page and clicking Panel Editor.

   If a panel has already been created but needs modifications, you may select the panel you would like to change from the Available Panel Tree and modify the existing panel accordingly. You can decide which files to use in the Sample File Tree. All files with a check are included in the panel, and files with a blank page are not selected. The selection or de-selection can be toggled by double clicking on the file or right clicking. Two different electropherogram modes can be toggled in panel manager, ‘Max & Average' mode and ‘Include All' mode. The toggle view button can also be used to toggle the Gel Image view in Panel Editor.

5. How do I create a new panel for my samples?

   To create a new panel either click the page or go to Tools >> Panel Editor. Click File >> Create New Panel in the toolbar. Enter a name for the new panel. You can select the specific panel type desired and method ("Automatic" and "Use All Samples" are suggested).

   Once the new panel has been created, you view the alleles in three different ways: Max & Average' mode, ‘Include All' mode, or gel image. These viewing modes can be toggled using the icon in the toolbar. If you need to add, delete, or edit an allele, right click on the specific location in the electropherogram or gel image (For a full description of editing alleles within the Panel Editor see GeneMarker Manual pages 16-18).

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4. Size Calibration Questions

1. Why do some of my samples fail during data processing?

   The Size Calibration function is designed to test the linearity of the size call for each lane. The size call fails when the quality of the sample is very low, because the allele peaks are not detected by the software. As a result, a linear graph cannot be created for samples where the allele peaks are undefined.

2. How do I correct for samples that failed to match the size standard?

   To correct for failed size calling, click the Size Calibration icon . Double click on the failed sample and add alleles to the electropherogram by right clicking on the center of each allele peak. Once you have added the alleles, right click anywhere on the electropherogram and select Update Calibration. Close this page and click the Call Allele icon . Select Call Allele-All Samples and click "OK."

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5. Data Report Questions

1. What does the "" symbol mean?

   The "" symbol means the allele call at that specific position is highly confident and does not need to be verified. You can set the "Pass" parameter within the "Additional Settings" box in the Run Wizard (See 2g).

2. What does the "" symbol ("OL") mean?

   The "" symbol means low confidence for this allele call, out of the possible allele number limit, 3 alleles in diploid species, or unmatched with the panels. "OL", Off-Ladder, means that the allele call at this position is out of range and therefore undetermined. For example, if a peak has a one or two base pair nucleotide difference from the standard peak, the software labels it as "OL," because the peak needs to be reviewed in the electropherogram for verification You are advised to check the electropherograms for alleles represented by this symbol for confirmation. This number can be set in the "Additional Settings" in the Run Wizard (See 5a).

   To save the reports, we suggest you to save them as text, htm files, then open in Excel, where it is possible to manipulate the report before printing.

3. What does the "" symbol mean?

   The "" symbol is used to represent allele peaks that are within the designated "Check" score range. You may set the allele score check range in the "Additional Settings" box in the Run Wizard (See 5a). Allele peaks within this range will be represented by this symbol and you are advised to verify these peaks in the electropherogram.

4. How do I save the allele list report?

   To save the allele list report, click the Save icon located above the allele report. The allele report will be saved as a text file, so that it can be imported into Excel for printing.

5. How do I print the allele list report?

   To print the allele report, save as a text file as described in 4e, and then open in an Excel spreadsheet in a delimited file format. Once you have opened the report in Excel, you can manipulate the columns and rows and print according to your specific needs.

6. How do I save the peak height information?

   To save the peak height information, click the Save Peak Table icon in the toolbar. The peak height information will be saved as a text file, which can opened and modified using Microsoft Word, Excel, etc.

7. Can I exclude the "OL" from being displayed in the allele report?

   You may prevent the " OL " symbol from being output to the report by simply erasing it from the report settings. Click the Report Settings icon and erase the Suspected Symbol field. When you save the Allele Report as a text file and import to Excel, only the Positive and Negative Symbols will be displayed.

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6. Printer Questions

1. How do I print to a PDF?

   In order to print to a PDF it is necessary to install Adobe Acrobat 5.0 in your computer. You may then go t o Print in GeneMarker and choose Acrobat Distiller from the drop-down list.

2. Is there a way for me to print comments added to the Peak Table?

   GeneMarker 1.2 does have this feature in the Print window. Click the Print Report icon located in the toolbar and then click the Advance button. Please check the option labeled "Print Project Comments."

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7. Pedigree Questions (Version 1.2 or Higher Only)

1. How do I link an existing pedigree file with the files in GeneMarker?

   After running the data in GeneMarker go to Tools>Pedigree Filename Match and open the existing pedigree file (.pre or .ped) in the first field. Next, load the sample files from the GeneMarker project into the second field. The family and individual identifiers are generally 14 to 18 and 19 to 23 respectively, but they may vary. Click " Process ," and a table will appear (. SMP file ) with the filenames correctly matched to the family and individual from the original pedigree file. Save this file in the same location as the original pedigree file. To open the linked pedigree file, go to Applications >Pedigree>Open Pedigree File and enter the original pedigree file in the top field. The linked .SMP file should automatically load into the second field.

2. Why are some of the pedigree nodes highlighted in red?

   The nodes highlighted in red have a marker with an allele/s that is inconsistent with Mendelian inheritance. In the image below, for example, child one is highlighted red, because she possesses allele 108 in marker Blue 3 when neither of her parents have this allele.

3. What does the question mark next to a node signify?

   The question mark signifies that one of the markers is empty for that sample.