GMT Tutorial Exercise 23 - Workflow 2 - Step 16

Import sample electrotraces into GeneMapper and determine peak sizes

1. Select File » Add Samples to Project from the file menu of the GeneMapper window. The Add Samples to Project dialogue box will apppear.




2. Browse for the ABI3730-generated folder with *.fsa files in the left-hand box. Select the sample files to be added to the project, and click the Add to List >> button. A folder containing the sample files to be added to the project appears in the right-hand box.
   
   Important:
   Every time a project is re-opened, GeneMapper has to be able to access the original *.fsa files in the same location as when they were first added to a project. Please consider carefully where *.fsa files will be most conveniently accessed in the future before adding samples to a project.
   




3. Click the Add button. The Add Samples to Project dialogue box will close and return you to the main GeneMapper window.




4. Select File » Save Project As... from the main menu. Enter a name for the project.
   The project name should be something meaningful and unique, such as the experiment name.
   Click OK.




5. Check the Analysis Method column. If the entries in this column are "None", click into the first cell under the header and select "Microsatellite Default" from the drop-down box that appears.
   Select the whole column by clicking on the column header, and use the key combination Ctrl + D to fill the whole column with this analysis method.




6. Open the "Microsatellite Default" analysis method by selecting Tools » GeneMapper Manager from the main menu.
   In the GeneMapper Manager window, click on the Analysis Method tab.
   Click on "Microsatellite Default" and click Open.
   In the Analysis Method Editor window that opens, click on the Allele tab.
   In the drop-down box at the top of this tab, select the "Bin Set"-setting: "None".




7. Check the Size Standard column. If this column is empty or displays "None", click in the first cell under the column header and select the size standard called "GS500LIZ_3730" from the drop-down box that appears.
   Select the whole column by clicking on the column header, and use the key combination Ctrl + D to fill the whole column with this size standard entry.




8. Select Analysis » Analyze from the main menu (or click the Analyze button: ).




9. GeneMapper will then determine peak sizes in the sample files. At the end of the analysis, the sample files will automatically be sorted by Sizing Quality (SQ) score, with suspect and failed samples appearing first.
   Samples passing the analysis are flagged with a green square, questionable samples are indicated by a yellow triangle, and failed samples are flagged with a red octagon.
   If you cannot see this data on the screen, you will need to use the scroll right (â–º) button, located at the bottom of the dialog box.
   
10. Marker data cannot be scored for sample files that are flagged by a red octagon. Depending on the reason for the failed capillary, it may be possible to override the Size Quality score:
    Click on the SQ flag for the failed sample.
    Select Analysis » Size Match Editor on the file menu. The Size Match Editor dialogue box appears.
    Press the Override SQ button in the dialogue box. If the sample data is beyond recovery then GeneMapper will provide an error message to prevent this operation from being performed.
    Note that while this operation often allows marker data to be scored for a failed capillary, allele size calling may not be completely accurate.