Identify Known Variants in One Sample (WES)

The Identify Known Variants in One Sample (WES) template workflow combines data analysis and interpretation. It should be used to identify known variants as specified by the user (e.g., known breast cancer associated variants) for their presence or absence in a sample. This workflow will not identify new variants.

The workflow maps the sequencing reads to a human genome sequence and does a local realignment of the mapped reads to improve the subsequent variant detection. In the next step, only variants specified by the user are identified and annotated in the newly generated read mapping.

Before starting the workflow, you may need to import your reads with the Import | Tracks tool (see

Run the Identify Known Variants in One Sample (WES) workflow

  1. Go to the toolbox and double-click on

            Template Workflows | Biomedical Workflows (Image biomedical_twf_folder_open_16_n_p) | Whole Exome Sequencing (Image exome_sequencing_closed_16_n_p) | General Workflows (WES) (Image general_wes_wf_closed_16_n_p) | Identify Known Variants from One Sample (WES) (Image identify_known_var_1_sample_wes_16_n_p)

  2. First select the reads of the sample that should be tested for presence or absence of your known variants (figure 20.11).

    Image identify_known_variants_in_one_sample_wizardstep1_wes
    Figure 20.11: 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.

  3. Next, specify the target regions (figure 20.12). 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.

    Image indels_target_knownwes
    Figure 20.12: Specify the target regions file.

  4. In the next wizard step, select the reference data set that should be used to identify the known variants (figure 20.13).

    Image identify_known_variants_in_one_sample_wes
    Figure 20.13: Choose the relevant reference Data Set to identify the known variants.

  5. Specify the parameters for the QC for Target Sequencing tool (figure 20.14).

    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.

    Image qc_settings_ikvwes
    Figure 20.14: 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.

  6. In the Identify Known Mutations from Mappings, select a variant track containing the known variants you want to identify in the sample (figure 20.15).

    Image identify_known_variants_in_one_sample_wizardstep4_wes
    Figure 20.15: 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).

  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 Finish.

Output from the Identify Known Variants in One Sample (WES)

The Identify Known Variants in One Sample (WES) tool produces five different output types:

It is a good idea to start looking at the Target Regions Coverage Report to see whether the coverage is sufficient in the regions of interest (e.g. > 30 ). Please also check that at least 90% of the reads are mapped to the human reference sequence. In case of a targeted experiment, we also recommend that you check that the majority of the reads are mapping to the targeted region.

When you have inspected the target regions coverage report you can open the Track List (see 20.16).

The Track List includes an overview track of the known variants and a detailed result track presented in the context of the human reference sequence, genes, transcripts, coding regions, targeted regions, and mapped sequencing reads.

Image identify_known_variants_in_one_sample_result1_wes
Figure 20.16: Track List that allows inspection of the identified variants in the context of the human genome and external databases.

Finally, a track with conservation scores has been added to be able to see the level of nucleotide conservation (from a multiple alignment with many vertebrates) in the region around each variant.

Open the annotated variant as a table showing all variants and the added information/annotations (see  20.17).

Image identify_known_variants_in_one_sample_result2_wes
Figure 20.17: Track List with an open overview variant track with information about if the variant has been detected or not, the identified zygosity, if the coverage was sufficient at this position and the observed allele frequency.

Note We do not recommend that any of the produced files are deleted individually as some of them are linked to other outputs. Please always delete all of them at the same time.