Detect QIAseq Methylation
The Detect QIAseq Methylation workflow can be used to call methylation levels on data generated with QIAseq Targeted Methyl Panels. Note that the workflow only supports Illumina paired-end sequencing data.
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, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=QC_Sequencing_Reads.html
- 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
- Reads are mapped using Map Bisulfite Reads to Reference, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Map_Bisulfite_Reads_Reference.html
- UMI reads are created using Calculate Unique Molecular Index Groups and Create UMI Reads from Grouped Reads
- Target coverage statistics are generated using QC for Targeted Sequencing, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=QC_Targeted_Sequencing.html
- Differential methylation is detected using Call Methylation Levels, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Call_Methylation_Levels.html
- Sample composition is optionally predicted using Predict Methylation Profile
- A summary report is created using Create Sample Report, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Create_Sample_Report.html
Launching the workflow
To run this workflow, go to:
Workflows | Template Workflows | Biomedical Workflows (
) | QIAseq Sample Analysis (
) | Other QIAseq workflows (
) | Detect QIAseq Methylation (
)
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 whether methylation profile prediction should be performed. Only select 'Yes' for data produced with T Cell Infiltration panels such as the 'T Cell Infiltration (MHS-202Z)' panel.
- 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 Methyl Panels hg19 Reference Data Set, see Reference data management for details.
Note that the QIAseq Targeted Methyl Panels are designed to use hg19 as reference, but custom panels can be designed against either hg19 or hg38. When analyzing data from a custom panel designed against hg38, a custom Reference Data Set must first be created, see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Reference_Data_Sets_defining_Custom_Sets.html and Import QIAGEN Primers 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.
- Target primers.
Select the primers corresponding to the panel used to generate the reads.
Specify the primers, either by selecting them from the Reference Data Set (QIAseq Methyl Panels hg19) for QIAGEN panels or, for custom panels, by importing them as described in Import QIAGEN Primers
- Target regions. Select the target regions that match the selected primers.
- Map Bisulfite Reads to Reference. Specify whether a 'Directional' (reads from both strands) or 'Non-directional' protocol was used to generate the data.
- QC for Target Sequencing. Specify the minimum coverage needed on all positions in a target for it to be considered covered. For somatic calling, we recommend setting this to at least 100x.
- Predict Methylation Profile. If you have selected to predict methylation profile, you will be asked to specify the minimum coverage a CpG site must have to be used in the prediction.
- 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 Targeted Methyl.
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