Identify QIAseq Multimodal DNA Lib Kit Variants
The following four workflows are designed to call somatic variants from DNA data produced with the QIAseq Multimodal DNA/RNA Lib Kit:
- Identify QIAseq Multimodal DNA Lib Kit and Hybrid Capture Somatic Variants (Illumina) for data generated with UMIs, followed by hybrid capture-based target enrichment without addition of mitochondrial spike-in probes, such as QIAseq Exome, QIAseq xHYB Human, or panels from a third party provider.
To analyze data generated with panels from a third party provider, see also QIAseq custom panels.
For data generated without UMIs, see Identify QIAseq Hybrid Capture DNA Somatic Variants (Illumina).
- Identify QIAseq Multimodal DNA Lib Kit and Hybrid Capture Somatic Variants with TMB and MSI (Illumina) (beta) for data generated with UMIs, followed by hybrid capture-based target enrichment, as described above.
The workflow furthermore calculates a TMB score and MSI status. TMB calculation is suitable for panels with sufficiently large target regions. Friends of Cancer Research recommends at least 667 kb [Vega et al., 2021]. The TMB evaluation is often accompanied by MSI assessment, as this can provide complementary information about the sample and possible treatment [Vanderwalde et al., 2018].
Please note that this workflow is in beta.
- Identify QIAseq Multimodal DNA Lib Kit with UMI Somatic Variants (WGS) (Illumina) for WGS data generated with UMIs.
- Identify QIAseq Multimodal DNA Lib Kit without UMI Somatic Variants (WGS) (Illumina) for WGS data generated without UMIs.
The workflows include all necessary steps for processing and analyzing the DNA reads:
- Various statistics summarizing and visualizing the input reads are produced using QC for Sequencing Reads, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=QC_Sequencing_Reads.html
- For data with UMIs, UMIs are removed using Remove and Annotate with Unique Molecular Index
- Reads are trimmed using Trim Reads, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Trim_Reads.html
- Reads are mapped using Map Reads to Reference, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Map_Reads_Reference.html
- For data with UMIs, UMI reads are created using Calculate Unique Molecular Index Groups and Create UMI Reads from Grouped Reads
- A guidance track is generated from the mapped (UMI) reads using Structural Variant Caller
- An improved mapping is obtained by realigning the mapped (UMI) reads using the guidance track and Local Realignment, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Local_Realignment.html
- For panel data, CNVs are optionally called from the improved mapping using Copy Number Variant Detection (CNVs). See https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Copy_Number_Variant_Detection.html
- Variants are called from the improved mapping using Low Frequency Variant Detection, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Low_Frequency_Variant_Detection.html. For panel data, variant calling is restricted to the relevant target regions.
- The variants are annotated with various information, such as the relation to repeat/homopolymer regions or gene elements, and are subsequently filtered to remove those that are likely to be artifacts through a filtering cascade using Filter on Custom Criteria, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Filter_on_Custom_Criteria.html
- For the workflow with TMB, the TMB score is determined using Calculate TMB Score
- For the workflow with MSI, the MSI status is determined using Detect MSI Status
- A summary report is created using Create Sample Report, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Create_Sample_Report.html
Launching the workflows
To run these workflows, go to
Template Workflows | Biomedical Workflows (
) | QIAseq Sample Analysis (
) | QIAseq DNA Workflows (
)
and select:
Identify QIAseq Multimodal DNA Lib Kit and Hybrid Capture Somatic Variants (Illumina) (
)
Identify QIAseq Multimodal DNA Lib Kit and Hybrid Capture Somatic Variants with TMB and MSI (Illumina) (beta) ()
Identify QIAseq Multimodal DNA Lib Kit with UMI Somatic Variants (WGS) (Illumina) ()
Identify QIAseq Multimodal DNA Lib Kit without UMI Somatic Variants (WGS) (Illumina) ()
For general information about launching workflows, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Launching_workflows_individually_in_batches.html
Options in the following dialogs can be configured for all four workflows:
- 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.
- Select Reads. Select the DNA reads. When analyzing more than one sample at a time, check the Batch checkbox in the lower left corner of the dialog.
- Select reference data set. Select the QIAseq Multimodal Library Kit and Hybrid Capture 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. See https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Running_workflows_in_batch_mode.html for details.
- Trim adapter lists. Select the trim adapter list with or without UMIs, as appropriate.
- 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.
- 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.
Options in the following dialogs can additionally be configured for the two hybrid capture workflows:
- Target regions/Select Target regions. Choose the relevant target regions. If the data is produced using a custom panel or a panel from a third party provider, see QIAseq custom panels. Note that for the workflow with TMB and MSI, the target regions are provided as a separate input, rather than being part of the Reference Data Set.
- Copy Number Variant Detection (CNVs). Choose Control mappings that are meaningful for the sample being analyzed, if copy number variants should be detected. See https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Copy_Number_Variant_Detection.html for details.
Options in the following dialogs can additionally be configured for the workflow with TMB and MSI:
- Detect MSI Status. Configure the options for MSI detection. Note that adjustments may be necessary. See Detect MSI Status for details.
- Calculate TMB Score. Configure the options for TMB score calculation. Note that adjustments may be necessary. See Calculate TMB Score for details.
Launching using the QIAseq Panel Analysis Assistant
The workflows are also available in the QIAseq Panel Analysis Assistant under Multimodal Library Kit, Exome, and xHYB Human.
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