How to run the Identify Known Variants in One Sample (WES) workflow

  1. Go to the toolbox and double-click on

            Toolbox | Ready-to-Use Workflows | Whole Exome Sequencing (Image exome_sequencing_closed_16_n_p) | General Workflows (WES) | Identify Known Variants from One Sample (WES) (Image identify_known_var_1_sample_wes_16_n_p)

  2. This will open the wizard step shown in figure 16.8 where you can select the reads of the sample that should be tested for presence or absence of your known variants.

    Image identify_known_variants_in_one_sample_wizardstep1_wes
    Figure 16.8: 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.

    Click on the button labeled Next.

  3. Specify the target region for the Indels and Structural Variants tool (figure 16.9). This step is optional and will speed the completion time of the workflow by running the tool only on the selected target regions. If you do not have a targeted region file to provide, simply click Next.

    Image indels_target_knownwes
    Figure 16.9: Specify the targeted region file for the Indels and Structural Variants tool.

  4. Specify the parameters for the QC for Target Sequencing tool (figure 16.10).

    When working with targeted data (WES or TAS data), quality checks for the targeted sequencing is 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.

    Image qc_settings_ikvwes
    Figure 16.10: 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.

    For more information about the tool, see QC for Target Sequencing.

  5. Click on the button labeled Next and specify the track with the known variants that should be identified in your sample (figure 16.11).

    Image identify_known_variants_in_one_sample_wizardstep4_wes
    Figure 16.11: 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).

    Click on the button labeled Next.

  6. In the last wizard step (figure 16.12)you can check the selected settings by clicking on the button labeled Preview All Parameters.

    Image identify_known_variants_in_one_sample_wizardstep7_wes
    Figure 16.12: Check the settings and save your results.

    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.

  7. Click on the button labeled OK to go back to the previous dialog box and choose to Save your results.

    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.