All peaks must have a bin in order to be analyzed using post-genotyping applications in ChimerMarker or GeneMarkerHID (chimerism testing, cell line authentication, mixture analysis, etc.). Sometimes, a sample will have a single peak that is not falling into a bin when all others are. This could be a microvariant, which is a partial repeat unit designated as a decimal (e.g.,14.2). To confirm an off-ladder peak is a microvariant allele, rather than an artifact, many labs will perform additional steps, such as:
- Confirming the expected microvariant is not in a stutter position or from spectral issues (e.g., pull up)
- Re-injecting or re-amplifying the sample to confirm the presence of the microvariant.
- If possible, the sample could be re-amplified using different primers to determine if the off-ladder allele is due to primer binding site mutations, rather than being a true microvariant.
- Consulting resources such as the NIST STRBase website to determine if the possible microvariant has been seen before
- If capillary electrophoresis results are ambiguous, sequencing can be performed to confirm the exact sequence.
If the laboratory determines the peak is a microvariant that should be included in the analysis, use the following steps to add the microvariant bin to the panel and re-analyze the data. Post genotyping applications cannot process peaks that are manually added if no bin for the allele is present in the panel.
Note: Figures below are from ChimerMarker software. The same steps are used to add a microvariant to a genotyping panel in GeneMarkerHID.
- Navigate to Tools – Panel Editor and click on the Project Panel, select Save As A New Panel from the File dropdown menu. Input the new panel name, click OK. The new panel will be located at the bottom of the Panel Templates list.
- Select the allelic ladder and samples, review the peaks, right mouse click on the microvariant peak, and select Insert Allele.
- Name the allele using an integer for the number of complete repeats followed by a decimal point and the number of bases in the partial repeat (e.g., 10.3 for a peak that is 3bp to the right of the 10 allele seen in the allelic ladder).
- Uncheck the Control Gene box to indicate this is a microvariant that is not seen in the allelic ladder.
- If this is a chimerism panel, edit the Origin (GeneMarkerHID does not have this field).
- Click Ok and then Save the edited panel. This panel will now be available in the Panel dropdown on the first screen of the Run Wizard for use in future analyses.
- You can right mouse click on the edited panel in the Panel Editor and select Set As Project Panel to reanalyze the samples with the updated panel without needing to go through the Run Wizard again.
Note: Only users with access rights to edit panels will be able to add bins to panels.