Analyze QIAseq RNA Fusion XP
The Analyze QIAseq RNA Fusion XP (Illumina) and the Analyze QIAseq RNA Fusion XP (Ion Torrent) template workflows can be used for analyzing data produced with the QIAseq RNA Fusion XP panels (JHS-001Z, JHS-002Z, JHS-003Z, JHS-004Z, JHS-005Z, JHS-3001Z, and JHS3002Z).
The panels allow fusion detection, variant calls, and expression values to be generated from the same RNA sample. This is possible due to the use of primers that have been annotated with the purpose for which they are intended: 'Variant', 'Fusion', and 'GEX'.
In the Analyze QIAseq RNA Fusion XP workflows, variants are called based on all reads while fusions are detected using reads matching 'Fusion'-annotated primers and gene expression is calculated based on reads matching 'GEX'-annotated primers.
The workflows include all necessary steps for processing and analyzing the reads:
- UMIs are removed using Remove and Annotate with Unique Molecular Index.
- Reads are trimmed using Trim Reads.
- UMI reads are created using Create UMI Reads from Reads.
- The reads are mapped using RNA-Seq Analysis.
- A QC report and primer coverage statistics for the mapped reads are created using QC for RNAscan Panels.
- Variants are called from the mapped reads 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.
- A summary report is created using Create Sample Report.
- A report is optionally prepared for uploading to QCI Interpret using Prepare QCI Interpret Upload.
- Specifically for 'Fusion' reads:
- 'Fusion' reads are extracted using Filter on Custom Criteria.
- Fusions are detected using Detect and Refine Fusion Genes.
- Specifically for 'GEX' reads:
- 'GEX' reads are extracted using Filter on Custom Criteria.
- Expression is quantified using RNA-Seq Analysis.
Launching the workflows
To run these workflows, go to
Workflows | Template Workflows | Biomedical Workflows (
) | QIAseq Sample Analysis (
) | QIAseq RNA Workflows (
)
and select:
Analyze QIAseq RNA Fusion XP (Illumina) (
)
Analyze QIAseq RNA Fusion XP (Ion Torrent) ()
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:
- Detect RNA variants. Skipping this may reduce workflow execution time.
- Quantify gene expression. Skipping this may reduce workflow execution time.
- Prepare for QCI Interpret. 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 RNA Fusion XP 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 primers. Choose the relevant primers from the drop down list.
- Detect and Refine Fusion Genes.
Configure the following options as needed:
- Detect exon skippings
- Detect novel exon boundaries
- Detect novel exon boundaries in both genes
- Gene filter action
- Genes for filtering (tracks)
- Fusion filter action
- Fusions for filtering (tables)
For details about the elements used by default in 'Genes for filtering (tracks)' and 'Fusions for filtering (tables)', see Exclude lists.
For general details about fusion detection, see Detect and Refine Fusion Genes.
- Filter on Custom Criteria, if running variant detection. Variant filters have 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.
- 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 workflows are also available in the QIAseq Panel Analysis Assistant under RNA Fusion XP.
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