Finding differentially methylated regions
The Targeted Methyl application of the Analyze QIAseq Samples guide offers a "Detect Differentially Methylated Regions" tool to be run after the Detect QIAseq Methylation workflow. The tool requires at least two case and two control samples.
Click Run to open the tool's wizard. In the first dialog, select the case read mappings, and click Next.
In the following dialog you can optionally choose to provide target regions. If these are supplied, then each target is separately tested for differential methylation. It makes sense to provide targets if they are the unit of biological interest, or if they are short enough that methylation patterns within a target are expected to be constant i.e. most Cs are hypomethylated/hypermethylated/unchanged when compared to a control sample. If no target regions are provided, the tool will search for differential methylation by dividing the genome into 1kb regions.
In the final dialog, select the control read mappings. The output of the tool is a track of differentially methylated regions for CpG sites.
The above-described tool works by calling the Call Methylation Levels tool (see http://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Call_Methylation_Levels.html) with parameters optimized for QIAseq Targeted Methyl data. To run the Call Methylation Levels tool directly with these optimized parameters:
- Uncheck Ignore duplicate matches: Duplicate matches have already been identified in the Detect QIAseq Methylation workflow using UMIs.
- Uncheck Confirm methylation contexts in reads: this option, when enabled, discards reads where the context is different in the reads and reference, for example due to a SNV. When this is disabled, SNVs that change the methylation context (for example from CpG to CHG) cannot be distinguished from changes in methylation. However this is unlikely to lead to incorrect interpretation of the data. The presence of a SNV can be confirmed by inspection in the read mapping.
- Set the Statistic mode to ANOVA
- Set the Minimum high-confidence site coverage to 10: Coverage should be high for most targets, so it may be acceptable to exclude low coverage sites which will typically be far from the primers, and which may add more noise than signal. However, first check that this is higher than the typical coverage of positions in each target using the Final_target_coverage output.
- Uncheck Create track of methylated cytosines: this would generate the same per-sample data already produced in the workflow.