Perform QIAseq Multimodal RNA Library Kit Analysis
RNA data produced with the QIAseq Multimodal DNA/RNA Library Kit can be analyzed using the following two template workflows:
- Perform QIAseq Multimodal RNA Library Kit Analysis (Illumina) for whole transcriptome sequencing data.
- Perform QIAseq Hybrid Capture RNA Analysis (Illumina) for data that has been subjected to hybrid capture-based target enrichment with the QIAseq xHYB CGP RNA Panel.
The workflows include all necessary steps for processing and analyzing the RNA reads:
- Various statistics summarizing and visualizing the input reads are produced using QC for Sequencing 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.
- Expression is quantified using RNA-Seq Analysis.
- Fusions are optionally detected using Detect and Refine Fusion Genes.
- A summary report is created using Create Sample Report.
The sensitivity of fusion detection is improved when RNA libraries are prepared using hybrid capture with Nextera adapter blockers.
Launching the workflows
To run these workflows, go to
Workflows | Template Workflows | Biomedical Workflows (
) | QIAseq Sample Analysis (
) | QIAseq RNA Workflows (
)
and select:
Perform QIAseq Multimodal RNA Library Kit Analysis (Illumina) (
)
Perform QIAseq Hybrid Capture RNA Analysis (Illumina) ()
For general information about launching workflows, see Launching workflows individually and in batches.
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.
- Specify workflow path. Select whether you want to detect fusions. Skipping fusion detection may speed up workflow execution time.
- 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 Multimodal RNA Library Kit and Hybrid Capture 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.
- Batch overview, if running in batch mode. Verify that the batching is as intended.
- Detect and Refine Fusion Genes, if running fusion detection.
Configure the following options as needed:
- Detect exon skippings
- Detect novel exon boundaries
- Detect novel exon boundaries in both genes
- For the Perform QIAseq Multimodal RNA Library Kit Analysis (Illumina) workflow:
- Gene filter action
- Genes for filtering (tracks)
- Fusion filter action
- Fusions for filtering (tables)
- For the Perform QIAseq Hybrid Capture RNA Analysis (Illumina) workflow:
- Fusion filter action
- Fusion for filtering (tables)
By default, the gene filtering options are set such that the workflow detects only fusions involving the genes targeted by the QIAseq xHYB CGP RNA Panel.
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.
- 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.
- 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 two workflows are also available in the QIAseq Panel Analysis Assistant. The Perform QIAseq Multimodal RNA Library Kit Analysis (Illumina) workflow is available under Multimodal Library Kit, and the Perform QIAseq Hybrid Capture RNA Analysis (Illumina) workflow is available under xHYB CGP.
Subsections