Identify Variants (WES-HD)

You can use the Identify Variants (WES-HD) ready-to-use workflow to call variants in the mapped and locally realigned reads. The workflow removes false positives and, in case of a targeted experiment, removes variants outside the targeted region. Variant calling is performed with the Fixed Ploidy Variant Detection tool.

The Identify Variants (WES-HD) ready-to-use workflow accepts sequencing reads as input.

Run the Identify Variants (WES-HD) workflow

To run the Identify Variants (WES-HD) workflow, go to:

        Toolbox | Ready-to-Use Workflows | Whole Exome Sequencing (Image exome_sequencing_closed_16_n_p) | Hereditary Disease (Image hereditary_folder_closed_16_n_p) | Identify Variants (WES-HD) (Image identify_variants_wes_16_h_p)

  1. Double-click on the Identify Variants (WES-HD) tool to start the analysis. If you are connected to a server, you will first be asked where you would like to run the analysis.

  2. Select the sequencing reads you want to analyze (figure 21.77).

    Image reads_iv_weshd
    Figure 21.77: Specify the sequencing reads for the appropriate family member.

  3. In the next dialog, you have to select which data set should be used to identify variants (figure 21.78).

    Image identify_variantshd_wes
    Figure 21.78: Choose the relevant reference Data Set to identify variants.

  4. Specify a target region file for the Indels and Structural Variants tool (figure 21.79).

    The targeted region file is a file that specifies which regions have been sequenced, when working with whole exome sequencing or targeted amplicon sequencing data. This file is something that you must provide yourself, as this file depends on the technology used for sequencing. You can obtain the targeted regions file from the vendor of your targeted sequencing reagents.

    Image indels_target_iv_weshd
    Figure 21.79: Specify the parameters for the Indels and Structural Variants tool.

  5. Specify the parameters for the Fixed Ploidy Variant Detection tool (figure 21.80).

    The parameters used by the Fixed Ploidy Variant Detection tool can be adjusted. We have optimized the parameters to the individual analyses, but you may want to tweak some of the parameters to fit your particular sequencing data. A good starting point could be to run an analysis with the default settings.

    Image fp_settings_iv_weshd
    Figure 21.80: Specify the parameters for the Fixed Ploidy Variant Detection tool.

    The parameters that can be set are:

    • Required variant probability is the minimum probability value of the 'variant site' required for the variant to be called. Note that it is not the minimum value of the probability of the individual variant. For the Fixed Ploidy Variant detector, if a variant site - and not the variant itself - passes the variant probability threshold, then the variant with the highest probability at that site will be reported even if the probability of that particular variant might be less than the threshold. For example if the required variant probability is set to 0.9 then the individual probability of the variant called might be less than 0.9 as long as the probability of the entire variant site is greater than 0.9.
    • Ignore broken pairs: When ticked, reads from broken pairs are ignored. Broken pairs may arise for a number of reasons, one being erroneous mapping of the reads. In general, variants based on broken pair reads are likely to be less reliable, so ignoring them may reduce the number of spurious variants called. However, broken pairs may also arise for biological reasons (e.g. due to structural variants) and if they are ignored some true variants may go undetected. Please note that ignored broken pair reads will not be considered for any non-specific match filters.
    • Minimum coverage: Only variants in regions covered by at least this many reads are called.
    • Minimum count: Only variants that are present in at least this many reads are called.
    • Minimum frequency: Only variants that are present at least at the specified frequency (calculated as 'count'/'coverage') are called.

  6. Specify the parameters for the QC for Targeted Sequencing tool, including a Target region file (figure 21.81).

    When working with targeted data (WES or TAS data), quality checks for the targeted sequencing is included in the workflows. Again, you can choose to use the default settings, or you can choose to adjust the parameters.

    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.

    Image qc_target_iv_weshd
    Figure 21.81: Specify the parameters for the QC for Targeted Sequencing tool.

  7. 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.

  8. Choose to Save your results and click on the button labeled Finish.

Output from the Identify Variants (WES-HD) workflow

The outputs generated are: