Perform QIAseq UPXome RNA Analysis
The Perform QIAseq UPXome RNA Analysis template workflow can be used for analyzing data produced with the QIAseq UPXome RNA Library Kits.
QIAseq UPXome RNA Library Kits can be used with the N6-T RT primer (random hexamer), the ODT-T RT primer (oligo-dT), or a combination of both. The ODT-T RT primer uses a TTTV tag to capture RNA fragments. The short polyT tag efficiently captures both exons and polyA tails, making it particularly advantageous for low-input samples or exosomes, where exon usage is of interest. This does not introduce a 3' bias, and approximately 50% of reads may map to exons, depending on the sample. While well-suited for differential expression analysis among samples with similar RNA input levels, it is not designed for RNA abundance analysis, as genes with more polyT regions tend to show higher expression levels.
The workflow contains a Demultiplex Reads element, but otherwise resembles the Perform QIAseq FastSelect RNA Analysis template workflow. This allows for simultaneous analysis of all samples in a multiplexed experiment. The workflow quantifies expression for each individual samples and subsequently performs different analyses across all samples. The ability to run parts of the workflow on a per-sample basis and other parts based on all samples, is possible due to the Iterate and Collect and Distribute elements, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Control_flow_elements.html
The workflow includes all necessary steps for processing and analyzing the reads:
- For each individual sample:
- The input reads are demultiplexed into individual samples using Demultiplex Reads, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Demultiplex_Reads.html
- Various statistics summarizing and visualizing the input reads are produced using QC for Sequencing Reads, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=QC_Sequencing_Reads.html
- Reads are trimmed using Trim Reads, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Trim_Reads.html
- Expression is quantified using RNA-Seq Analysis, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=RNA_Seq_Analysis.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
- Additionally, if using the N6-T RT primer:
- N6-T RT primer reads are extracted using Trim Reads and a trim adapter list to remove reads containing the ODT-T RT primer, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Trim_Reads.html
- Fusions are optionally detected using Detect and Refine Fusion Genes, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Detect_Refine_Fusion_Genes.html
- Across all samples:
- Principal Component Analysis (PCA) plots are created using PCA for RNA-Seq, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=PCA_RNA_Seq.html
- Expression browsers are created using Create Expression Browser, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Create_Expression_Browser.html
- A combined report is created using Combine Reports, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Combine_Reports.html
Launching the workflow
To run this workflow, go to:
Workflows | Template Workflows | Biomedical Workflows (
) | QIAseq Sample Analysis (
) | QIAseq RNA Workflows (
) | Perform QIAseq UPXome RNA Analysis (
)
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.
- Specify workflow path.
Select the primers used to produce the data.
Select whether you want to detect fusions (for the N6-T RT primer only). Skipping fusion detection may speed up workflow execution time.
- Select Reads. Select the input reads.
- Specify reference data handling. Select the QIAseq UPXome and FastSelect RNA hg38 Reference Data Set, see Reference data management for details.
- Demultiplex Reads. Specify the barcodes, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Demultiplex_Reads.html for details. This can for example be done by loading a barcode table generated by Detect Wells for UPXome.
- 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
- 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.
- 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 UPXome RNA.
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