QIAseq xHYB CGP FFPE Fastq to Somatic Variants
The QIAseq xHYB CGP FFPE Fastq to Somatic Variants template workflow supports analysis of data generated from FFPE with QIAseq xHYB CGP DNA panel both stand-alone and with selected spike-ins. Three panels are supported:
- QIAseq xHYB CGP DNA (target regions: QIAseq_xHYB_CGP_DNA_Panel_target_regions_v1.0)
- QIAseq xHYB DNA Fusion & MSI+ (target regions: QIAseq_xHYB_CGP_DNA_Fusion_MSI_Panel_target_regions_v1.0)
- QIAseq xHYB CGP HRD (target regions: QIAseq_xHYB_CGP_HRD_target_regions_v1.0)
It can be used to:
- Identify and annotate somatic variants and generate various QC metrics.
- Identify copy number variants (CNV) if control coverage tables are provided.
- Assess homologous recombination deficiency (HRD) if QIAseq xHYB CGP and QIAseq xHYB HRD panels are used in combination.
- Detect fusions.
- Detect micro satellite instability (MSI).
- Calculate tumor mutational burden (TMB).
- Generate control coverage tables for CNV detection.
For all panels, CNV detection is performed on the QIAseq_xHYB_CGP_DNA_Panel_target_regions_v1.0 regions only.
The workflow can be found at:
Template Workflows | LightSpeed Workflows (
) | QIAseq Workflows (
) | QIAseq xHYB (
) | QIAseq xHYB CGP FFPE Fastq to Somatic Variants (
)
Options in the following dialogs can be configured:
- Choose where to run If you are connected to a CLC Server via your 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 the workflow path The following paths can be configured:
- Analysis options Specify whether you want to
- Analyze sample Detect variants and, optionally perform additional analyses described below.
- Create CNV control Create a CNV control coverage table from a normal sample.
- Analyze sample and create CNV control Detect variants and create a CNV control coverage table.
- Detect CNVs
- Yes CNVs will be called if control coverage tables are provided in the Copy Number Variant Detection (Targeted) dialog.
- No CNVs will not be called.
- Detect fusions
- Yes Fusions will be called.
- No Fusions will not be called.
- Detect MSI
- Yes MSI will be detected.
- No MSI will not be detected.
- Calculate TMB
- Yes TMB will be calculated.
- No TMB will not be calculated.
- Prepare for QCI Interpret
- Yes A report that prepares the sample analysis results for later upload to QCI Interpret will be created from the relevant tracks and reports produced by the analysis.
- No No QCI Interpret report will be produced.
- Assess HRD
- Yes HRD will be assessed. Note that this should only be carried out when the QIAseq xHYB CGP and QIAseq xHYB HRD panels are used in combination.
- No HRD will not be assessed.
- Analysis options Specify whether you want to
- Specify reference data handling Select the relevant Reference Data Set. QIAseq DNA xHYB CGP hg38 will be pre-selected and is recommended for running this workflow. If you have not downloaded the Reference Data Set yet, the dialog will offer the opportunity to download it using the Download to Workbench button. If the QIAseq DNA xHYB CGP hg38 reference data set does not contain the needed target regions, a custom reference data set can be created.
- LightSpeed Fastq to Somatic Variants
Specify options for the LightSpeed Fastq to Somatic Variants tool (for a detailed description of the tool, see Lightspeed Fastq to Somatic Variants):
- Reads (fastq) Press Browse to select fastq files for analysis.
- Minimum frequency (%) Specify the minimum variant allelle frequency for detected variants.
- SNV minimum average quality Specify the minimum average quality of detected SNVs.
- Indel minimum average quality Specify the minimum average quality of detected indels.
- Batch Select if fastq files from different samples are used as input, and each sample should be analyzed individually (for information about batching see Batching).
- Join lanes when batching Select to join fastq files from the same sample that were sequenced on different lanes.
- Target regions Choose the relevant target regions from the drop down list.
- If MSI detection is enabled, MSI baseline. Specify the MSI baseline which the sample will be compared to, in order to detect micro satellite instability. The Detect MSI Status tool measures the statistical variation of the length distribution of each micro satellite locus and decides for each locus if it is stable or not by comparing the statistical variation of the test sample with the normal samples' baseline. If the proportion of unstable micro satellite loci is higher than a predefined threshold, then the sample is considered unstable.
We recommend that the MSI baseline used is created using micro satellite stable (MSS) samples that are sequenced under the same lab conditions as the sample for which the MSI status is calculated. Baselines can be created using the Generate MSI Baseline tool. To use your created MSI baseline in analysis, open a copy of the workflow, and remove the workflow role from the MSI baseline input. Specify your MSI baseline and save the workflow, or leave the MSI baseline input channel unlocked, and specify your baseline when you run the workflow.
MSI baselines for the QIAseq xHYB CGP DNA and QIAseq xHYB DNA Fusion & MSI+ panels are provided for demonstration purposes (QIAseq_xHYB_CGP_msi_baseline_v1.0 and QIAseq_xHYB_CGP_Fusion_MSI_baseline_demo_v1.0). Analysis of QIAseq xHYB CGP HRD data should be carried out with QIAseq_xHYB_CGP_msi_baseline_v1.0.
- If CNV detection is enabled, CNV controls. Specify Controls against which the coverage pattern in your sample will be compared in order to call CNVs. If you do not supply any controls, the CNV analysis will not be carried out. Please note that if you want the CNV analysis to be done, it is important that the controls supplied are meaningful controls for the sample being analyzed. Coverage tables of control samples to be used in the CNV analysis can be created by running this workflow with Analysis options set to Create CNV control or Analyze sample and create CNV control in the dialog Specify workflow path.
A meaningful control must satisfy two conditions: (1) It must have a copy number status that is meaningful to compare against. For panels with targets on the X and Y chromosomes, the control and sample should be matched for gender. (2) The control read mapping must result from the same type of processing that will be applied to the sample. For more information about CNV detection, see Copy Number Variant Detection.
To use your own CNV controls in analysis, open a copy of the workflow, and remove the workflow role from the CNV control input. Specify your controls and save the workflow, or leave the Controls input channel unlocked, and specify your controls when you run the workflow.
CNV controls for the QIAseq xHYB CGP DNA, QIAseq xHYB DNA Fusion & MSI+, and QIAseq xHYB CGP HRD panels are provided for demonstration purposes (QIAseq_xHYB_CGP_CNV_v1.0, QIAseq_xHYB_CGP_Fusion_MSI_CNV_demo_v1.0, and QIAseq_xHYB_CGP_HRD_CNV_v1.0).
Note that targets on X and Y chromosomes have been removed from the demo CNV controls and CNV detection will therefore not be carried out on X and Y chromosomes when using these.
- QC for Targeted Sequencing Set the Minimum coverage parameter of the QC for Targeted Sequencing tool. Using default settings, samples where 90 percent of target region positions do not meet this threshold will be flagged in the sample report generated by the workflow.
- If fusion detection is enabled, Filter Fusions on Support and Filter Fusions on Gene Distance and Direction. Fusion 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 Detect Fusion Genes from DNA for details.
- Filter Variants 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.
A low-confidence filter has been included in this workflow to reduce errors commonly associated with FFPE samples. Pronounced deamination in FFPE material results in the LightSpeed variant-caller model assigning lower confidence to affected nucleotide changes. By applying a stringent significance threshold to low-frequency variants, the workflow effectively reduces FFPE-related artifacts in samples with typical FFPE error profiles.
- If TMB calculation is enabled, Calculate TMB Score. Specify whether you want the report to provide a TMB status based on thresholds (see Calculate TMB Score (LightSpeed) for details). Only reports with a TMB status can be uploaded to QCI Interpret (see Prepare QCI Interpret Upload).
- If preparing a QCI Interpret report, Prepare QCI Interpret Upload. Specify sample name, subject ID, and project.
- 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. For additional information, see Create Sample Report.
- 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.
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