Analyze QIAseq Hybrid Capture DNA
The Analyze QIAseq Hybrid Capture DNA template workflows can be used for analyzing DNA reads generated using the QIAseq Multimodal DNA Library Kit (without UMIs) or QIAseq FX DNA Library Kit followed by hybrid capture-based target enrichment, such as QIAseq Exome, QIAseq xHYB Human, or panels from a third party provider. For panels from a third party provider, the same approach as described in QIAseq custom panels is recommended.
The workflow can analyze samples with or without mitochondrial spike-in probes.
The following workflows are available:
- Analyze QIAseq Hybrid Capture DNA Germline (Illumina)
- Analyze QIAseq Hybrid Capture DNA Somatic (Illumina)
The workflows include all necessary steps for processing and analyzing the reads:
- Various statistics summarizing and visualizing the input reads are produced using QC for Sequencing Reads.
- Target coverage statistics are generated using QC for Targeted Sequencing.
- Reads are trimmed using Trim Reads.
- Reads are mapped to the human reference genome using Map Reads to Reference.
- 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.
- Variants are detected in the relevant target regions from the improved mapping using Low Frequency Variant Detection for somatic workflows and Fixed Ploidy Variant Detection for germline workflows.
- 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.
- For somatic workflows, a report is optionally prepared for uploading to QCI Interpret using Prepare QCI Interpret Upload.
Both workflows optionally detect and filter mitochondrial variants. CNVs are not detected for mitochondrial target regions.
Launching the workflows
To run the workflows, go to:
Workflows | Template Workflows | Biomedical Workflows (
) | QIAseq Sample Analysis (
) | QIAseq DNA workflows (
) | Analyze QIAseq Hybrid Capture DNA Germline/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:
- Include mitochondrial. Specify whether the sample is sequenced with mitochondrial spike-in probes.
- 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, for somatic workflows. Prepare a report that can later be uploaded to QCI Interpret or QCI Interpret Translational.
- 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 Hybrid Capture and WGS 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.
- 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.
- Low Frequency Variant Detection or Fixed Ploidy Variant Detection. Configure the Minimum frequency (%) as needed, if variants should be detected at lower frequencies. Note that the value should be no more than half of the frequency specified when filtering variants.
- 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.
If mitochondrial analysis is included, the same options as described above can also be configured in the following dialogs:
- QC for Targeted Sequencing (mito)
- Low Frequency Variant Detection (mito)
- Filter on Custom Criteria (mito)
Launching using the QIAseq Panel Analysis Assistant
The workflows are also available in the QIAseq Panel Analysis Assistant under Human Exome and xHYB Human.
Subsections