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Genotyping Project
Genotyping Projects are used to determine the alleles present in unknown samples. When combined with only a Bin Estimator, functionality is similar to Genemapper, allowing for rapid parameterized peak identification and binning that can be manually reviewed. When also combined with an Artifact Estimator, peak identification becomes much more nuanced, allowing for much improved artifact peak identification and resolution of low density signal.
To create a new Genotyping Project, select the Genotyping Projects tab in the left panel. Fill in the relevant information, including a unique title, the locus set to be analyzed, a valid Bin Estimator Project that will be used to bin peaks, optionally an Artifact Estimator Project to aid in identifying artifact peaks, and also optionally a Quantification Bias Estimator Project to get a more accurate relative quantification of alleles, allowing for potential phasing of haplotypes in mixed populations.
After creating a Genotyping Project, navigate to the Samples tab and select from the inactive samples the samples you wish to incorporate into the project. After selecting, presss Add to add them to the project. Alternatively, you may upload a CSV file with a header of [Barcode] and each row is the unique barcode of the sample to be analyzed. The sample must exist in the database prior to this, such as when samples are added during Plate Map assignment.
After creating a new Genotyping Project and adding Samples, navigate to the Analysis Settings tab. From here, each locus may be individually analyzed to determine the alleles present in each sample at the selected locus.
| Parameter | Description |
|---|---|
| Min Relative Peak Height | Peaks with a height relative to the tallest peak that do not exceed Min Relative Peak Height are classified as artifact |
| Min Absolute Peak Height | Minimum peak height for a peak to not be classified as artifact |
| Bleedthrough Limit | Maximum bleedthrough ratio before a peak is classified as bleedthrough |
| Crosstalk Limit | Maximum crosstalk ratio before a peak is classified as crosstalk |
| Failure Threshold | At least one peak's height must exceed the failure threshold, otherwise the run is deemed a failure and rejected |
| Soft Artifact SD Limit | Peaks with a height that falls within the Soft Artifact SD Limit times the artifact error plus the estimated artifact contribution have a peak probability calculated when probabilistic annotation is enabled |
| Hard Artifact SD Limit | Peaks with a height minus (artifact contribution + artifact error * Hard Artifact SD Limit) < Min Absolute Peak Height are classified as artifact |
| Genotyping Probability Threshold | Peaks with a probability less than the Genotyping Probability Threshold are classified as artifact |
| Bootstrap Probability Threshold | Peaks with a probability less than the Bootstrap Probability Threshold are culled during the iterative peak frequency and sample MOI estimation steps during probabilistic peak annotation |
| Offscale Threshold | Runs with a peak that exceeds the Offscale Threshold are flagged as offscale and are avoided if possible |
After analysis, you may view individual sample genotypes by navigating to the Genotypes Viewer tab. To view a genotype, select a sample from the list, which will then populate the panel to the right with the locus specific results. Select a locus from the right panel to view the identified genotype. Orange bars indicate a called allele.