Analyze QIAseq DNA Ultra Somatic
The Analyze QIAseq DNA Ultra Somatic (Illumina) template workflow can be used for analyzing DNA reads generated using a QIAseq Ultra panel. These panels are designed to provide high coverage in targeted regions to allow identification of low frequency variants in cfDNA.
The workflow is configured to detect very low frequency variants. Please note that to call very low frequency variants, coverage must be high. In low coverage samples or regions, very low frequency variants are unlikely to be represented in the 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.
- 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 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 DNA Ultra Somatic (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).
- 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 DNA Ultra Panels 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 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.
- Target primers. Choose the relevant primers from the drop down list.
- 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.
- Create UMI Reads from Grouped Reads. Specify settings for UMI grouping. QIAseq Ultra data is expected to contain very large UMI groups and more PCR or sequencing errors may consequently be present in the UMIs compared to other sequencing protocols. Therefore, settings for grouping reads into UMI groups should be more relaxed than settings for standard panels. This is reflected in the default settings for Create UMI Reads from Grouped Reads.
- 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.
- 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 Targeted DNA Ultra.
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