Perform QIAseq RNA Fusion XP Analysis
The Perform QIAseq RNA Fusion XP Analysis (Illumina) and the Perform QIAseq RNA Fusion XP Analysis (Ion Torrent) 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, and therefore improve on the capabilities of the existing QIAseq RNAscan panels. These extended capabilities are supported by primers which have been annotated with the purpose for which they are intended: 'Variant', 'Fusion', and 'GEX'.
In the Perform QIAseq RNA Fusion XP Analysis workflows, variants are called based on all reads while fusions are detected using reads matching 'Fusion'-annotated primers and gene expressions are 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, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Trim_Reads.html
- UMI reads are created using Create UMI Reads from Reads
- The reads are mapped using RNA-Seq Analysis, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=RNA_Seq_Analysis.html
- Various statistics summarizing the mapped reads are produced using QC for RNAscan Panels
- Variants are called from the mapped reads using Low Frequency Variant Detection, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Low_Frequency_Variant_Detection.html
- 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, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Filter_on_Custom_Criteria.html
- A summary report is created using Create Sample Report, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Create_Sample_Report.html
- Specifically for 'Fusion' reads:
- 'Fusion reads are extracted using Filter on Custom Criteria, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Filter_on_Custom_Criteria.html
- The reads are mapped using RNA-Seq Analysis, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=RNA_Seq_Analysis.html
- Various statistics summarizing the mapped reads are produced using QC for RNAscan Panels
- Fusions are detected using Detect and Refine Fusion Genes, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Detect_Refine_Fusion_Genes.html
- Specifically for 'GEX' reads:
- 'GEX' reads are extracted using Filter on Custom Criteria, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Filter_on_Custom_Criteria.html
- Expression is quantified using RNA-Seq Analysis, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=RNA_Seq_Analysis.html
Launching the workflows
To run these workflows, go to
Workflows | Template Workflows | Biomedical Workflows (
) | QIAseq Sample Analysis (
) | QIAseq RNA Workflows (
)
and select:
Perform QIAseq RNA Fusion XP Analysis (Illumina) (
)
Perform QIAseq RNA Fusion XP Analysis (Ion Torrent) ()
For general information about launching workflows, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Launching_workflows_individually_in_batches.html
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. 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, you will be asked to define the batch units. See https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Running_workflows_in_batch_mode.html for details.
- Batch overview, if running in batch mode. Verify that the batching is as intended.
- RNA primers.
Select the primers that were used to produce the data.
The workflow requires the panel primers to be specified, either by selecting them from the Reference Data Set (QIAseq RNA Fusion XP Panels hg38) for QIAGEN panels or, for custom panels, by importing them as described in Import QIAGEN Primers.
- Identify candidate variants. The filtering cascade has been configured to provide the best sensitivity and precision in the output variants. The cascade has been tuned using samples of relatively high quality and coverage. Therefore, additional filtering might be needed, or filtering values adjusted when working with low quality/coverage samples. See https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Filter_on_Custom_Criteria.html for details on how to adjust the options.
- Remove False Positives (filter on allele frequency). Optionally adjust the minimum frequency of detected variants.
- 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 https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Exclude_lists.html.
For general details about fusion detection, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Detect_Refine_Fusion_Genes.html.
- Create Sample Report. Select relevant summary items and specify thresholds for quality control. Summary items, thresholds, and an indication of whether specified thresholds were met, will be shown in the quality control section of the sample report. The default summary items are appropriate for many data sets, but may need to be adjusted. See https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Create_Sample_Report.html for details.
- 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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