- Go to the toolbox and double-click on the "Identify Known Variants from One Sample" ready-to-use workflow
This will open the wizard step shown in figure 14.37 where you can select the reads of the sample, which should be tested for presence or absence of your known variants.
Select all sequencing reads from your sample. If several samples should be analyzed, the tool has to be run in batch mode. This is done by selecting "Batch" (tick "Batch" at the bottom of the wizard as shown in figure 14.37) and select the folder that holds the data you wish to analyse. If you have your sequencing data in separate folders, you should choose to run the analysis in batch mode.
When you have selected the sample(s) you wish to analyze, click on the button labeled Next. In the following wizard steps, you will be asked to provide the target regions track a number of times as this is relevant for different analyses within the ready-to-use workflow.
- In this wizard step you can select your target regions track to be used for calling of InDels and structural variants (figure 14.38).
- In the next wizard step you can select your target regions track and specify the minimum coverage to be used when checking the quality of the targeted sequencing. The minimum coverage will be used to provide the length of each target region that has at least this coverage. You can also specify whether or not to ignore non-specific matches and broken pairs. When these are applied, reads that are non-specifically mapped or belong to broken pairs will be ignored (figure 14.39).
- Click on the button labeled Next and in specify the track with the known variants that should be identified in your sample (figure 14.40). Furthermore, in this wizard step you can specify the minimum read coverage for the position of the variant that should be identified. If the coverage at the position of the variant is below this, the result will show this.
The parameter "Detection Frequency" will be used in the calculation twice. First, it will report in the result if a variant has been detected (observed frequency > specified frequency) or not (observed frequency <= specified frequency). Moreover, it will determine if a variant should be labeled as heterozygous (frequency of another allele identified at a position of a variant in the alignment > specified frequency) or homozygous (frequency of all other alleles identified at a position of a variant in the alignment < specified frequency).
- Click on the button labeled Next, which will take you to the next wizard step (figure 14.41). In
this and the next dialog, you will be asked about which of the annotations/informations added to variants should be included in the results.
Please specify your track with known variants.
- Click on the button labeled Next and once again select the same track with known variants (figure 14.42).
- Click on the button labeled Next to go to the last wizard step (figure 14.43).
In this wizard step you can check the selected settings by clicking on the button labeled Preview All Parameters. In the Preview All Parameters wizard you can only check the settings, it is not possible to make any changes at this point. At the bottom of this wizard there are two buttons regarding export functions; one button allows specification of the export format, and the other button (the one labeled "Export Parameters") allows specification of the export destination. When selecting an export location, you will export the analysis parameter settings that were specified for this specific experiment.
- Click on the button labeled OK to go back to the previous dialog box and choose Save.
Note! If you choose to open the results, the results will not be saved automatically. You can always save the results at a later point.