Identify QIAseq Multimodal DNA Library Kit Variants
Three workflows are designed to call somatic variants from DNA data with UMIs produced with the QIAseq Multimodal DNA/RNA Library Kit.
DNA data without UMIs produced with the QIAseq Multimodal DNA/RNA Library Kit, can be analyzed using the Hybrid Capture template workflows Identify QIAseq Hybrid Capture DNA Germline Variants (Illumina) (see Identify QIAseq Hybrid Capture DNA Germline Variants (Illumina)) and Identify QIAseq Hybrid Capture DNA Somatic Variants (Illumina) (see Identify QIAseq Hybrid Capture DNA Somatic Variants (Illumina)) as well as the Identify QIAseq Somatic Variants (WGS) (Illumina) template workflow (see Identify QIAseq Somatic Variants (WGS) (Illumina)).
The three available workflows for data with UMIs are:
- Identify QIAseq Hybrid Capture Somatic Variants (with UMI) (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.
- Identify QIAseq Hybrid Capture DNA Somatic Variants with TMB and MSI (with UMI) (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 Library Kit with UMI Somatic Variants (WGS) (Illumina) for WGS data generated with 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
- 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
- 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 (Targeted). 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 Hybrid Capture DNA Somatic Variants (with UMI) (Illumina) (
)
Identify QIAseq Hybrid Capture DNA Somatic Variants with TMB and MSI (with UMI) (Illumina) (beta) ()
Identify QIAseq Multimodal DNA Library Kit with 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 three 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.
- Specify reference data handling. 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.
- Identify candidate variants. The filtering cascade has been tuned using samples of relatively high quality and coverage to provide the best possible sensitivity and precision. Additional filtering may be needed, or filtering values may need to be adjusted, when working with low quality/coverage samples or when seeking a different balance between sensitivity and precision. 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.
- QC for Target Sequencing. Set the Minimum coverage parameter of the QC for Target Sequencing tool. Using default settings, samples where 90 percent of target region positions do not meet this threshold will be flagged in the sample report generated by the workflow.
- Copy Number Variant Detection (Targeted). Choose Controls that are meaningful for the sample being analyzed, if copy number variants should be detected. The template workflow Create QIAseq Hybrid Capture CNV Control Mapping (with UMI) (Illumina) can be used to generate the controls (see Create QIAseq Hybrid Capture CNV Control Mapping (with UMI) (Illumina)). For more information about copy number variant detection see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Copy_Number_Variant_Detection.html.
- Create Sample Report. Specify QC items for assessment and subsequent flagging in the sample report generated by the workflow. For additional information, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Create_Sample_Report.html.
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 Identify QIAseq Multimodal DNA Library Kit with UMI Somatic Variants (WGS) (Illumina) template workflow is available in the QIAseq Panel Analysis Assistant under the Multimodal Library Kit.
The Identify QIAseq Hybrid Capture DNA Somatic Variants (with UMI) (Illumina) and Identify QIAseq Hybrid Capture DNA Somatic Variants with TMB and MSI (with UMI) (Illumina) (beta) template workflows are not available in the Panel Analysis Assistant.
Subsections
- Output from the Identify QIAseq Multimodal DNA Library Kit Variants workflows
- Create QIAseq Hybrid Capture CNV Control Mapping (with UMI) (Illumina)