Analyze QIAseq Multimodal DNA Panel
The Analyze QIAseq Multimodal DNA Panel (Illumina) template workflow can be used for analyzing DNA reads generated using QIAseq Multimodal Panels. Analyze QIAseq Multimodal RNA Panel can be used for RNA reads.
The workflow includes all necessary steps for processing and analyzing the reads:
- Target coverage statistics are generated using QC for Targeted Sequencing.
- UMIs are removed using Remove and Annotate with Unique Molecular Index.
- Reads are trimmed using Trim Reads.
- Reads are mapped to the human reference genome 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 reads using Structural Variant Caller.
- An improved mapping is obtained by realigning the mapped reads using the guidance track and Local Realignment.
- Primers are trimmed in the improved mapping using Trim Primers of Mapped Reads.
- Variants are detected in the relevant target regions from the improved mapping using Low Frequency Variant Detection.
- Variants that most likely originate from RNA contamination are identified and filtered using Annotate RNA Variants.
- The variants are annotated with various information, such as overlap with repeat/homopolymer regions and genes, and are subsequently filtered to remove likely artifacts 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.
- A report is optionally prepared for uploading to QCI Interpret using Prepare QCI Interpret Upload.
Launching the workflow
To run this workflow, go to:
Workflows | Template Workflows | Biomedical Workflows (
) | QIAseq Sample Analysis (
) | QIAseq DNA Workflows () | Analyze QIAseq Multimodal DNA Panel (Illumina) (
)
See Launching workflows individually and in batches for general information.
Options can be configured in the following wizard steps:
- 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. The following paths can be configured:
- Analysis Options. Specify whether to:
- Analyze sample: Detect variants and optionally perform additional analyses described below.
- Create CNV control: Create coverage table from a normal sample.
- Analyze sample and create CNV control: Perform both.
- Detect CNVs. Detect copy number variants. This requires CNV controls (i.e. normal samples).
- Calculate TMB. Calculate a tumor mutation burden score. This is only recommended for panels covering at least 667 kb [Vega et al., 2021].
- Detect MSI. Calculate a microsattelite instability status. This requires an MSI baseline generated from normal samples.
- Prepare for QCI Interpret. Prepare a report that can later be uploaded to QCI Interpret or QCI Interpret Translational.
- Analysis Options. Specify whether to:
- Select Reads. Select the input reads. When analyzing more than one sample at a time, check Batch in the lower left corner of the dialog.
- Specify reference data handling. Select the QIAseq Multimodal DNA Panels hg38 or QIAseq Multimodal Pan Cancer DNA Panel hg38 Reference Data Set, see Reference data management for details.
- Configure batching, if running the workflow in batch mode. Define the batch units.
- Batch overview, if running the workflow in batch mode. Verify that the batching is as intended.
- Target primers. Choose the relevant primers from the drop down list.
- Target regions. Choose the relevant target regions from the drop down list. If the data was produced using a panel that is not available in the list, see QIAseq custom panels.
- Mispriming events. Optionally select a mispriming events track to improve primer trimming. The track can be generated using Identify Mispriming Events.
- MSI baseline, if detecting MSI.
Select an MSI baseline. If none is provided, MSI detection will not be performed.
For best results, the baseline should match the sample in key experimental parameters, such as sequencing technology. For more details and a list of baselines available in the Reference Data Manager, see Detect MSI Status.
- Map Reads to Reference. Configure masking. By default, regions defined by the Genome Reference Consortium are excluded to remove false duplications, including one involving U2AF1.
- QC for Targeted Sequencing. Configure the Minimum coverage.
- Copy Number Variant Detection (Targeted), if detecting CNVs.
Specify Controls for detecting CNVs. If none are provided, CNV detection will not be performed.
For best results, the controls should match the sample in key experimental parameters, such as sequencing technology and, when targets include regions on the X and/or Y chromosomes, gender. For more details, see Copy Number Variant Detection.
- Filter on Custom Criteria. Configure how variants should be filtered. The default options are optimized for samples with relatively high quality and coverage providing a strong balance between sensitivity and precision. For lower-quality or lower-coverage samples, when seeking a different sensitivity-precision balance, or for detecting low-frequency variants, additional filters or adjusted thresholds might be needed. See Filter on Custom Criteria for details on how to configure the options.
- Calculate TMB Score, if calculating a TMB score. Specify whether the report should provide a TMB status based on thresholds, see Calculate TMB Score for more details. Only reports containing a TMB status can be uploaded to QCI Interpret.
- Create Sample Report. Select relevant summary items and configure thresholds for quality control. These are included in the quality control section of the sample report. The default values are appropriate for most data sets, but may need to be adjusted.
- Prepare QCI Interpret Upload, if preparing a QCI Interpret report. Specify sample name, subject ID, and project.
- 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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