Identify Variants (TAS)

The Identify Variants (TAS) tool takes sequencing reads as input and returns identified variants as part of a Track List.

The tool runs an internal workflow, which starts with mapping the sequencing reads to the human reference sequence. Then it runs a local realignment to improve the variant detection, which is run afterwards.

Two different variant callers are used; the Low Frequency Variant Detection tool is used to call small insertions, deletions, SNVs, MNV, and replacements, and the InDel and Structural Variants tool calls larger insertions, deletions, translocations, and replacements. By the end of the variant detection, variants that have been detected by the Low Frequency Variant Detection tool with an average base quality smaller than 20 are filtered away.

In addition, a targeted region report is created to inspect the overall coverage and mapping specificity in the targeted regions.

Before starting the workflow, you will need to import in the workbench a file with the genomic regions targeted by the amplicon or hybridization kit. Such a file (a BED or GFF file) is usually available from the vendor of the enrichment kit and sequencing machine. Use the Import | Tracks tool to import it in your Navigation Area.

Run the Identify Variants (TAS) workflow

To run the Identify Variants (TAS) workflow, go to:

        Ready-to-Use Workflows | Targeted Amplicon Sequencing (Image targeted_sequencing_closed_16_n_p) | Somatic Cancer (Image somatic_folder_closed_16_n_p) | Identify Variants (TAS (Image identify_variants_tas_16_n_p)

  1. Select the sequencing reads from the sample that should be analyzed (figure 18.28).

    Image identify_variants_wizardstep1_tas
    Figure 18.28: Please select all sequencing reads from the sample to be analyzed.

    If several samples should be analyzed, the tool has to be run in batch mode. This is done by checking "Batch" and selecting the folder that holds the data you wish to analyze.

  2. In the next dialog, you have to select which data set should be used to identify variants (figure 18.29).

    Image identify_variants_tas
    Figure 18.29: Choose the relevant reference Data Set to identify variants in your sample.

  3. In the Indels and Structural Variants dialog you can restrict calling of such variants to the targeted regions (figure 18.30).

    Image identify_variants_wizardstep2_tas
    Figure 18.30: Select the track with the targeted regions from your experiment.

  4. In the next wizard step (figure 18.31) you can specify the parameters for variant detection.

    Image identify_variants_wizardstep3_tas
    Figure 18.31: Specify the parameters for variant detection.

  5. In the next wizard step (figure 18.32) you have to specify the track with the targeted regions from the experiment. You can also specify the minimum read coverage, which should be present in the targeted regions.

    Image identify_variants_wizardstep4_tas
    Figure 18.32: Select the track with the targeted regions from your experiment.

  6. 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.

  7. Choose to Save your results and click Finish.

Output from the Identify Variants (TAS) workflow

The Identify Variants (TAS) tool produces six different types of output:

It is important that you do not delete any of the produced files individually as some of the outputs are linked to other outputs. If you would like to delete the outputs, please always delete all of them at the same time.

First have a look at the mapping report to see if the coverage is sufficient in regions of interest (e.g. > 30 ). Furthermore, check that at least 90% of reads are mapped to the human reference sequence. In case of a targeted experiment, also check that the majority of reads are mapping to the targeted region.

Afterwards please open the Track List file (see  18.33).

The Track List includes the track of identified variants in context to the human reference sequence, genes, transcripts, coding regions, targeted regions and mapped sequencing reads.

Image identify_variants_result1_tas
Figure 18.33: The Track List allows you to inspect the identified variants in the context of the human genome.

Open the variant track as a table to see information about all identified variants (see 18.34).

Image identify_variants_result2_tas
Figure 18.34: Track List with an open track table to inspect identified variants more closely in the context of the human genome.