Many assays multiplex 5-26 loci in 4-6 dye channels for each sample; examples include Trisomy/Aneuploidy, and Microsatellite Instability (MSI).
Other assays only use 1 or 2 dyes, often with overlapping loci in the same dye, so they split their loci into separate tubes. In addition to the overlapping loci regions which require separate PCR reactions, they also require separate panels to evaluate all of the loci. Examples include Cystic Fibrosis (CFTR) and B-Cell Clonality.
The following two examples can be applied to the many commercially available kits and custom lab developed chemistries that fall into this category.
Example one: Devyser CFTR 68 Data Analysis Workflow
(Devyser CFTR 68 CE-IVD, 7-A044-EN, version 2021-09-13)
The Devyser CFTR 68 kit uses two different mixes to amplify all the necessary loci for the assay. The data generated from these two mixes will need to be analyzed using separate panels in GeneMarker. Please reference the user manual chapter 5 creating custom panels, analysis corner webinar Creating Panels in GeneMarker software, or contact tech_support@softgenetics.com for assistance in creating panels.
The steps below can be used for any test that requires two different PCR mixes.
- Use the same naming convention for files from Mix 1 and Mix 2. In order to group files correctly, the same file name sections must be used consistently for both the sample IDs and the designators for CFTR mix 1 or 2. In the example below, the file name convention is CFTR68-Mix#-Sample#-POP4.fsa.
- Open two instances of GeneMarker. Import the Mix 1 samples in one instance and the Mix 2 samples in the second, and analyze them with the appropriate analysis templates and panels.
- Compare the results for the ID1 and ID2 markers from the Mix 1 files to the appropriate Mix 2 files to confirm the replicates have the same genotype. The figure below shows this step in the main analysis screen. If there are a large number of samples, selecting the dye channel with the ID markers in the Project Comparison tool is the most efficient way to automatically identify any mis-matched sample pairs (Chapter 8, Additional Tools Project Comparison in the linked Help menu or user manual pdf).
Note: Any sample pairs that did not have the same genotype for the two ID markers should be disabled before merging results.
- If electropherograms are required, save each project, export and collate the allele reports, selecting the desired fields (e.g., electropherograms, report table, report header). Report header and electropherograms were selected in the example below.
- Alternatively, if electropherogram images are not required, the Merge Project tool in GeneMarker can be used to merge the result tables for a complete report in one table (Details in Chapter 8 in the linked help or Genotype Merge Application Note).
Example 2: Invivoscribe IGH + IGK B-Cell Clonality Assay Workflow
(Invivoscribe 280314 rev. H October 2023)
This kit consists of five separate amplification tubes for each sample - three for IGH and two for IGK. Since there is no overlap of marker ranges for the IGH tubes, many labs validate pooling the IGH tubes. This allows analysis using one panel for all the IGH targets. Products of the IGK amplifications have overlapping loci in the same dye, so a panel is needed for IGK tube A and another panel for IGK tube B. Once again, please reference the user manual chapter 5 creating custom panels, analysis corner webinar Creating Panels in GeneMarker software, or contact tech_support@softgenetics.com for assistance in creating panels.
This workflow is very similar to example 1, except there will be three-to-five sets of data, each analyzed in their own project, with their own panels. Analyze the raw data (.fsa, .scf, .hid) files with the appropriate panel, save the projects and allele reports, and collate the allele reports for IGH and IGK for each sample.
If desired, the Merge Project tool in GeneMarker can be used to merge the result tables of the three-to-five projects for a complete report in one table (Chapter 8 in the linked help or Genotype Merge Application Note).