Run the Identify Known Variants in One Sample (WES) workflow
- To run the Identify Known Variants in One Sample (WES) template workflow, go to:
Workflows | Template Workflows | Biomedical Workflows (
) | Whole Exome Sequencing (
) | General Workflows (WES) (
) | Identify Known Variants from One Sample (WES) (
)
- First select the reads of the sample that should be tested for presence or absence
of your known variants (figure 19.11).
Figure 19.11: Select the sequencing reads from the sample you would like to test for your known variants.If several samples from different folders should be analyzed, the tool has to be run in batch mode. This is done by selecting "Batch" and specifying the folders that hold the data you wish to analyse.
- Next, specify the target regions (figure 19.12). The target regions are used by the tools Structural Variant Caller and QC for Targeted Sequencing. Providing the Structural Variant Caller with target regions, reduces the processing time of the workflow as the tool only analyzes defined target regions.
Figure 19.12: Specify the target regions file. - In the next wizard step, select the reference data set that should be used to identify the known variants (figure 19.13).
Figure 19.13: Choose the relevant reference Data Set to identify the known variants. - Specify the parameters for the QC for Target Sequencing tool (figure 19.14).
When working with targeted data (WES or TAS data), quality checks for the targeted sequencing are included in the workflows. This step is not optional, and you need to specify the targeted regions file adapted to the sequencing technology you used. Choose to use the default settings or to adjust the parameters.
Figure 19.14: Specify the parameters for the QC for Target Sequencing tool.The parameters that can be set are:
- Minimum coverage provides the length of each target region that has at least this coverage.
- Ignore non-specific matches reads that are non-specifically mapped will be ignored.
- Ignore broken pairs reads that belong to broken pairs will be ignored.
- In the Identify Known Mutations from Mappings, select a variant track containing the known variants you want to identify in the sample (figure 19.15).
Figure 19.15: Specify the track with the known variants that should be identified.The parameters that can be set are:
- Minimum coverage The minimum number of reads that covers the position of the variant, which is required to set "Sufficient Coverage" to YES.
- Detection frequency The minimum allele frequency that is required to annotate a variant as being present in the sample. The same threshold will also be used to determine if a variant is homozygous or heterozygous. In case the most frequent alternative allele at the position of the considered variant has a frequency of less than this value, the zygosity of the considered variant will be reported as being homozygous.
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).
- In the last 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, and if you wish to make changes you have to use the Previous button from the wizard to edit parameters in the relevant windows.
- Choose to Save your results and click Finish.