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• Introduction
  • The concept of Biomedical Genomics Analysis plugin
  • System requirements
  • Contact information
• QIAseq Panel Analysis
  • Data management
  • The Analyze QIAseq Panels guide
    • Import your reads
    • Start a QIAseq panel analysis
  • How to create a custom panel analysis workflow
    • Import QIAGEN Primers
  • Create a custom Trim adapter list (optional)
• Targeted DNA
  • The Identify QIAseq DNA Variants ready-to-use workflows
    • Output from the Identify QIAseq DNA Variants workflow
    • Quality Control for the Identify QIAseq DNA Variants workflow
  • Targeted DNA tools detailed description
    • Remove and Annotate with Unique Molecular Index
    • Calculate Unique Molecular Index Groups
    • Create UMI Reads
    • Remove Ligation Artifacts
    • Trim Primers of Mapped Reads
    • Annotate Variants with Unique Molecular Index Info
    • Prepare Guidance Variant Track
• Targeted RNAscan
  • The Detect QIAseq RNAscan Fusions ready-to-use workflow
    • Output from the Detect QIAseq RNAscan Fusions workflow
    • Interpretation of the Detect QIAseq RNAscan Fusions results
  • Targeted RNAscan tools detailed description
    • Detect Fusion Genes
    • Refine Fusion Genes
    • Annotate Fusions with Known Fusion Information
    • QC for RNAscan Panels
    • Import Known Fusion Information Track
• Targeted RNA
  • The Quantify QIAseq RNA Expression ready-to-use workflow
    • Output from the Quantify QIAseq RNA Expression workflow
  • Targeted RNA tools detailed description
    • The Quantify QIAseq RNA tool
• QCI Interpret Integration
  • Configuration of the Upload to QCI Interpret tool
  • The Upload to QCI Interpret tool
• QIAGEN GeneRead DNA Panel Analysis
  • The QIAGEN GeneRead Panel Analysis workflow
    • Output from the QIAGEN GeneRead Panel Analysis
  • Trim Primers and their Dimers of Mapped Reads
• Ready-to-use workflows descriptions and guidelines
  • General Workflow
  • Somatic Cancer
  • Hereditary Disease
• Getting started
  • Import sequencing data
  • Prepare Raw Data
• Whole genome sequencing (WGS)
  • General Workflows (WGS)
    • Annotate Variants (WGS)
    • Identify Known Variants in One Sample (WGS)
  • Somatic Cancer (WGS)
    • Filter Somatic Variants (WGS)
    • Identify Somatic Variants from Tumor Normal Pair (WGS)
    • Identify Variants (WGS)
  • Hereditary Disease (WGS)
    • Filter Causal Variants (WGS-HD)
    • Identify Causal Inherited Variants in Family of Four (WGS)
    • Identify Causal Inherited Variants in Trio (WGS)
    • Identify Rare Disease Causing Mutations in Family of Four (WGS)
    • Identify Rare Disease Causing Mutations in Trio (WGS)
    • Identify Variants (WGS-HD)
• Whole exome sequencing (WES)
  • General Workflows (WES)
    • Annotate Variants (WES)
    • Identify Known Variants in One Sample (WES)
  • Somatic Cancer (WES)
    • Filter Somatic Variants (WES)
    • Identify Somatic Variants from Tumor Normal Pair (WES)
    • Identify Variants (WES)
    • Identify and Annotate Variants (WES)
  • Hereditary Disease (WES)
    • Filter Causal Variants (WES-HD)
    • Identify Causal Inherited Variants in Family of Four (WES)
    • Identify Causal Inherited Variants in Trio (WES)
    • Identify Rare Disease Causing Mutations in Family of Four (WES)
    • Identify Rare Disease Causing Mutations in Trio (WES)
    • Identify Variants (WES-HD)
    • Identify and Annotate Variants (WES-HD)
• Targeted amplicon sequencing (TAS)
  • General Workflows (TAS)
    • Annotate Variants (TAS)
    • Identify Known Variants in One Sample (TAS)
  • Somatic Cancer (TAS)
    • Filter Somatic Variants (TAS)
    • Identify Somatic Variants from Tumor Normal Pair (TAS)
    • Identify Variants (TAS)
    • Identify and Annotate Variants (TAS)
  • Hereditary Disease (TAS)
    • Filter Causal Variants (TAS-HD)
    • Identify Causal Inherited Variants in Family of Four (TAS)
    • Identify Causal Inherited Variants in Trio (TAS)
    • Identify Rare Disease Causing Mutations in Family of Four (TAS)
    • Identify Rare Disease Causing Mutations in Trio (TAS)
    • Identify Variants (TAS-HD)
    • Identify and Annotate Variants (TAS-HD)
• Whole Transcriptome Sequencing (WTS)
  • Annotate Variants (WTS)
  • Compare Variants in DNA and RNA
  • Identify Candidate Variants and Genes from Tumor Normal Pair
  • Identify Variants and Add Expression Values
  • Identify and Annotate Differentially Expressed Genes and Pathways
• Batching workflows
• Appendices
  • Legacy tools
    • Trim Primers of Mapped Single Reads
    • Trim Primers of Mapped Paired End Reads
  • Install and uninstall plugins
    • Install
    • Uninstall
• Bibliography

Subsections

• Run the Identify Somatic Variants from Tumor Normal Pair (TAS) workflow
• Output from the Identify Somatic Variants from Tumor Normal Pair (TAS) workflow

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Identify Somatic Variants from Tumor Normal Pair (TAS)

The Identify Somatic Variants from Tumor Normal Pair (TAS) ready-to-use workflow can be used to identify potential somatic variants in a tumor sample when you also have a normal/control sample from the same individual.

When running this workflow the reads are mapped and the variants identified. An internal workflow removes germline variants that are found in the mapped reads of the normal/control sample and variants outside the target region are removed as they are likely to be false positives due to non-specific mapping of sequencing reads. Next, remaining variants are annotated with gene names, amino acid changes, conservation scores and information from relevant databases like ClinVar (variants with clinically relevant association). Finally, information from dbSNP is added to see which of the detected variants have been observed before and which are completely new.

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 Somatic Variants from Tumor Normal Pair (TAS) workflow

To run the Identify Somatic Variants from Tumor Normal Pair (TAS) tool, go to:

        Toolbox | Ready-to-Use Workflows | Targeted Amplicon Sequencing | Somatic Cancer () | Identify Somatic Variants from Tumor Normal Pair (TAS) ()

1. Go to the toolbox and double-click on the Identify Somatic Variants from Tumor Normal Pair (TAS) ready-to-use workflow.

2. First, (figure 12.21), select the tumor sample reads.

   
   Figure 12.21: Select the tumor sample reads.

3. In the next wizard step, specify the normal sample reads.

4. In the next dialog, select which data set should be used to identify variants (figure 12.22).

   
   Figure 12.22: Choose the relevant reference Data Set to identify variants.

5. The following 2 steps allow you to restrict the calling of Indels and Structural Variants to the targeted regions, both for tumor and normal reads (figure 12.23).

   
   Figure 12.23: Specify the target regions track.

6. Set the parameters for the Low Frequency Variant Detection step (figure 12.24).

   
   Figure 12.24: Specify the settings for the variant detection.

   For a description of the different parameters that can be adjusted, see http://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Low_Frequency_Variant_Detection.html. If you click on "Locked Settings", you will be able to see all parameters used for variant detection in the ready-to-use workflow.

7. In the following 2 wizard steps, you can select your target regions track to be used for reporting the performance of the targeted re-sequencing experiment for the tumor and normal samples successively (figure 12.25). The targeted region track should be the same as the track you selected in the previous wizard steps. Variants found outside the targeted regions will not be included in the output that is generated with the ready-to-use workflow.

   
   Figure 12.25: Select your target region track.

   For a description of the different parameters that can be adjusted, see http://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=QC_Targeted_Sequencing.html. If you click on "Locked Settings", you will be able to see all parameters used for the QC for Targeted Sequencing tool in the ready-to-use workflow.

8. In the Remove Variants Present in Control Reads step, you can adjust the settings for removal of germline variants (figure 12.26).

   
   Figure 12.26: Specify setting for removal of germline variants.

9. 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.

10. Choose to Save your results and click Finish.

Output from the Identify Somatic Variants from Tumor Normal Pair (TAS) workflow

The following outputs are generated:

• Read Mapping Normal () The mapped sequencing reads for the normal sample. The reads are shown in different colors depending on their orientation, whether they are single reads or paired reads, and whether they map unambiguously (see http://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Coloring_mapped_reads.html).
• Read Mapping Tumor () The mapped sequencing reads for the tumor sample.
• Target Region Coverage Report Normal () The report consists of a number of tables and graphs that in different ways provide information about the mapped reads from the normal sample.
• Target Region Coverage Tumor () A track showing the targeted regions. The table view provides information about the targeted regions such as target region length, coverage, regions without coverage, and GC content.
• Target Region Coverage Report Tumor () The report consists of a number of tables and graphs that in different ways provide information about the mapped reads from the tumor sample.
• Amino Acids Changes Track that shows the consequences of the variants at the amino acid level in the context of the original amino acid sequence. A variant introducing a stop mutation is illustrated with a red amino acid.
• Variants () A variant track holding the identified variants that are found in the targeted regions. The variants can be shown in track format or in table format. When holding the mouse over the detected variants in the Track List a tooltip appears with information about the individual variants. You will have to zoom in on the variants to be able to see the detailed tooltip.
• Annotated Somatic Variants () A variant track holding the identified and annotated somatic variants. The variants can be shown in track format or in table format. When holding the mouse over the detected variants in the Track List, a tooltip appears with information about the individual variants. You will have to zoom in on the variants to be able to see the detailed tooltip.
• Track List Tumor Normal Comparison () A collection of tracks presented together. Shows the annotated variants track together with the human reference sequence, genes, transcripts, coding regions, the mapped reads for both normal and tumor, the annotated somatic variants, information from the ClinVar database, and finally a track showing the conservation score (see figure 12.27).

Figure 12.27: The Track List presents all the different data tracks together and makes it easy to compare different tracks.

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