Analyze QIAseq FastSelect and UPXome RNA
The Analyze QIAseq FastSelect and UPXome RNA (Illumina) template workflow can be used for analyzing data produced with the QIAseq FastSelect RNA Library Kits and QIAseq UPXome RNA Library Kits.
These kits can be used with the ODT-T RT primer (oligo-dT), N6-T RT primer (random hexamer), 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 quantifies expression for each individual sample and for UPXome 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 Control flow elements.
The workflow includes all necessary steps for processing and analyzing the reads:
- Various statistics summarizing and visualizing the input reads are produced using QC for Sequencing Reads.
- Reads are trimmed using Trim Reads.
- Expression is quantified using RNA-Seq Analysis.
- A summary report is created using Create Sample Report.
- A report is optionally prepared for uploading to QCI Interpret using Prepare QCI Interpret Upload.
- Additionally, if using the N6-T RT primer:
- N6-T RT primer reads are extracted using Trim Reads with a trim adapter list to remove reads containing the ODT-T RT primer.
- Fusions are optionally detected using Detect and Refine Fusion Genes.
- Additionally, if analyzing UPXome samples:
- The input reads are demultiplexed into individual samples using Demultiplex Reads.
- Principal Component Analysis (PCA) plots are created using PCA for RNA-Seq.
- Expression browsers are created using Create Expression Browser.
- A combined report is created using Combine Reports.
Launching the workflow
To run this workflow, go to:
Workflows | Template Workflows | Biomedical Workflows (
) | QIAseq Sample Analysis (
) | QIAseq RNA Workflows (
) | Analyze QIAseq FastSelect and UPXome RNA (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:
- Analysis options. Specify the kit used to produce the data (FastSelect or UPXome). Select UPXome demultiplexing only for demultiplexing reads produced with UPXome, without further analysis.
- Primer(s) used. Select the primers used to produce the data.
- Detect fusions. Select whether to detect fusions (for the N6-T RT primer only). 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 UPXome and FastSelect RNA 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.
- Demultiplex Reads (for UPXome only). Specify the barcodes, see Demultiplex Reads 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 detecting fusions. 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.
- 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 workflow is also available in the QIAseq Panel Analysis Assistant under DNA/RNA Library Kit.
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