Perform QIAseq Multimodal Panel Analysis

The Perform QIAseq Multimodal Panel Analysis (Illumina) template workflow can be used for analyzing DNA and/or RNA reads generated using QIAseq Multimodal Panels.

Note that the QIAseq Multimodal Panels are designed against genome build hg19 for the DNA panel and hg38 for the RNA panel. BED files are provided in the respective genome build. However, the template workflow requires that reference data for both DNA and RNA is for the same genome build. The two QIAseq Multimodal Reference Data Sets provided by the Reference Data Manager are for genome build hg38, where the reference data for the DNA panel has been converted to hg38 as described below.

For custom panels, the DNA panel BED file needs to be imported against hg19, after which it should be converted to hg38 using the tool Convert Annotation Track Coordinates. If many regions are lost during conversion, it can cause reads to be discarded that would have otherwise mapped to the lost target regions. To avoid such issues, a copy of the template workflow can be used, containing only the analysis of the DNA reads, and the workflow should be run using the imported BED file against hg19.

The workflow is built by combining variant calling from the Identify QIAseq DNA Somatic Variants (Illumina) workflow, and fusion detection from the Perform QIAseq RNAscan Fusion XP workflow, with some minor adjustments. Specifically, two tools to further annotate variants have been added:

Annotate RNA Variants
Annotate with Repeat and Homopolymer Information, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Annotate_with_Repeat_Homopolymer_Information.html

The annotations added by these tools are used to filter away variant calls that most likely originate from RNA contamination and variants appearing within repeat or homopolymer regions.

The workflow can be run with the Reference Data Set QIAseq Multimodal Panels hg38. This set contains Catalog Panel Primers and Target Regions that have been lifted to the hg38 reference sequence. You can either download the reference data set before starting the analysis or download the default data set during execution of the workflow.

Launching the workflow

To run this workflow, go to:

For general information about launching workflows, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Launching_workflows_individually_in_batches.html

Options can be configured in the following dialogs:

Choose where to run. If you are connected to a CLC Server via the CLC Workbench, you will be asked where you would like to run the analysis. We recommend that you run the analysis on a CLC Server when possible.
Specify workflow path. Select whether you want to analyze DNA and/or RNA reads.
Select DNA/RNA Reads. Select the reads. When analyzing more than one sample at a time, check the Batch checkbox in the lower left corner of the dialog.
Specify reference data handling. Select the QIAseq Multimodal Panels hg38 Reference Data Set, see Reference data management for details.
Configure batching. If running the workflow in Batch mode, you will be asked to define the batch units.
Batch overview, if running in batch mode. Verify that the batching is as intended.
DNA/RNA primers. Select the primers that were used to produce the data.
Target regions. Select the target regions that match the selected primers.
Map Reads to Reference. Configure masking.
By default, the GenomeReferenceConsortium_masking_hg38_no_alt_analysis_set masking track is selected, containing the regions defined by the Genome Reference Consortium, which serve primarily to remove false duplications, including one affecting the gene U2AF1. Changing the masking mode from 'No masking' to 'Exclude annotated' excludes these regions.
QC for Target Sequencing. Specify the minimum coverage needed on all positions in a target for it to be considered covered. For somatic calling, we recommend setting this to at least 100x
Copy Number Variant Detection (Targeted). Specify a control mapping against which the coverage pattern in your sample will be compared in order to call CNVs. If you do not specify a control mapping, or if the target regions files contains fewer than 50 regions, the Copy Number Variation analysis will not be carried out.
Create Sample Report (DNA/RNA). Select relevant summary items and specify thresholds for quality control. Summary items, thresholds, and an indication of whether specified thresholds were met, will be shown in the quality control section of the sample report. The default summary items are appropriate for many data sets, but may need to be adjusted.
Identify candidate variants. The filtering cascade has been configured to provide the best sensitivity and precision in the output variants. The cascade has been tuned using samples of relatively high quality and coverage. Therefore, additional filtering might be needed, or filtering values adjusted when working with low quality/coverage samples. See https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Filter_on_Custom_Criteria.html for details on how to adjust the options.
Remove False Positives (Filter on allele frequency). Specify the minimum frequency of detected variants.
Add Information about Amino Acid Changes. Leave the genetic code set to 1 Standard.
Detect and Refine Fusion Genes. Configure the following options as needed:
- Detect exon skippings
- Detect novel exon boundaries
- Detect novel exon boundaries in both genes
- Gene filter action
- Genes for filtering (tracks)
- Fusion filter action
- Fusions for filtering (tables)
For details about the elements used by default in 'Genes for filtering (tracks)' and 'Fusions for filtering (tables)', see Exclude lists.
For general details about fusion detection, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Detect_Refine_Fusion_Genes.html.
Result handling. Choose if a workflow result metadata and/or log should be saved.
Save location for new elements. Choose where to save the data, and press Finish to start the analysis.

Launching using the QIAseq Panel Analysis Assistant

The workflow is also available in the QIAseq Panel Analysis Assistant under Multimodal Panels.

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