Identify QIAseq Multimodal DNA Library Kit Variants
DNA data without UMIs produced with the QIAseq Multimodal DNA/RNA Library Kit, can be analyzed using the following template workflows, described elsewhere in the manual:
- Identify QIAseq Hybrid Capture DNA Germline Variants (Illumina), see Identify QIAseq Hybrid Capture DNA Germline Variants (Illumina).
- Identify QIAseq Hybrid Capture DNA Somatic Variants (Illumina), see Identify QIAseq Hybrid Capture DNA Somatic Variants (Illumina).
- Identify QIAseq Somatic Variants (WGS) (Illumina) template workflow, see Identify QIAseq Somatic Variants (WGS) (Illumina).
DNA data with UMIs produced with the QIAseq Multimodal DNA/RNA Library Kit can be analyzed using the following two template workflows, which are designed to call somatic variants:
- Identify QIAseq Multimodal DNA Library Kit with UMI Somatic Variants (WGS) (Illumina) for WGS data.
- Identify QIAseq Hybrid Capture DNA Somatic Variants (with UMI) (Illumina) for data that has been subjected to hybrid capture-based target enrichment without addition of mitochondrial spike-in probes such as QIAseq Exome, QIAseq xHYB Human, QIAseq xHYB CGP DNA, or panels from a third-party provider.
The workflow optionally detects CNVs, calculates a TMB score, and detects MSI status.
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.
- UMIs are removed using Remove and Annotate with Unique Molecular Index.
- Reads are trimmed using Trim Reads.
- Reads are mapped using Map Reads to Reference.
- 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.
- Variants are called from the improved mapping using Low Frequency Variant Detection. 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.
- CNVs are optionally detected from the improved mapping using Copy Number Variant Detection (Targeted).
- A TMB score is optionally calculated using Calculate TMB Score.
- MSI status is optionally detected using Detect MSI Status.
- A summary report is created using Create Sample Report.
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 Library Kit with UMI Somatic Variants (WGS) (Illumina) (
)
Identify QIAseq Hybrid Capture DNA Somatic Variants (with UMI) (Illumina) ()
For general information about launching workflows, see Launching workflows individually and in batches.
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.
- Select Reads. Select the input 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.
For the Identify QIAseq Multimodal DNA Library Kit with UMI Somatic Variants (WGS) (Illumina) workflow:
- Select the QIAseq Multimodal Library Kit and Hybrid Capture hg38 Reference Data Set.
For the Identify QIAseq Hybrid Capture DNA Somatic Variants (with UMI) (Illumina) workflow:
- Select the QIAseq Multimodal Library Kit and Hybrid Capture hg38 Reference Data Set if the data was hybrid captured using the QIAseq Exome or QIAseq xHYB Human panels.
- Select the QIAseq DNA xHYB CGP hg38 Reference Data Set if the data was hybrid captured using the QIAseq xHYB CGP DNA panel.
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.
- 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 Filter on Custom Criteria 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 Identify QIAseq Multimodal DNA Library Kit with UMI Somatic Variants (WGS) (Illumina) workflow:
- Map Reads to Reference. In the Map Reads to Reference dialog, it is possible to configure masking. A custom masking track can be used, but by default, the masking track is set to GenomeReferenceConsortium_masking_hg38_no_alt_analysis_set, containing the regions defined by the Genome Reference Consortium, which serve primarily to remove false duplications, including one affecting the gene U2AF1.
Options in the following dialogs can additionally be configured for the Identify QIAseq Hybrid Capture DNA Somatic Variants (with UMI) (Illumina) workflow:
- Specify workflow path.
Select whether you want to:
- Detect CNVs. Note that CNV detection requires CNV controls (i.e. normal samples).
If you select "Yes", you must further specify whether the CNV controls contain targets on X and Y:
- Select Yes (X and Y not in controls) if there are no target regions on X and Y in the CNV controls.
- Select Yes (X and Y in controls) if there are target regions on X and Y in the CNV controls and you are analyzing a sample from the same sex as the CNV controls. This will enable detection of CNVs on the X and Y chromosomes.
For the QIAseq xHYB CGP DNA panel, a CNV coverage table based on 13 different normal samples without X and Y regions is available in the Reference Data Manager.
- Detect MSI. Note that MSI detection requires an MSI baseline.
For the QIAseq xHYB CGP DNA panel, an MSI baseline based on 30 normal samples and 176 loci from msisensor2 is available in the Reference Data Manager.
- Calculate TMB. Note that TMB calculation is only recommended for panels covering at least 667 kb [Vega et al., 2021].
- Detect CNVs. Note that CNV detection requires CNV controls (i.e. normal samples).
- 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.
- 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.
- Calculate TMB Score. Configure the options for TMB score calculation. See Calculate TMB Score for details.
- Create Sample Report. Specify QC items for assessment and subsequent flagging in the sample report generated by the workflow. For additional information, see Create Sample Report.
Launching using the QIAseq Panel Analysis Assistant
The two workflows are also available in the QIAseq Panel Analysis Assistant. The Identify QIAseq Multimodal DNA Library Kit with UMI Somatic Variants (WGS) (Illumina) template workflow is available under Multimodal Library Kit, and the Identify QIAseq Hybrid Capture DNA Somatic Variants (with UMI) (Illumina) template workflow is available under xHYB CGP.
Subsections
- Output from the Identify QIAseq Multimodal DNA Library Kit Variants workflows
- Create QIAseq Hybrid Capture CNV Control Mapping (with UMI) (Illumina)