5-base Methylation QC

The 5-base methylation QC task in the Connected Multiomics enables you to visualize sample-level QC metrics that describe reads mapping quality and CpG methylation calling. The QC metrics are extracted from the DRAGEN analysis metric files that were ingested into the study as required files for 5-base DNA Prep data analysis in the Connected Multiomics. To invoke the 5-base methylation QC task:

• At Analyses page, click on the 5-base Methylation node.

• Click QA/QC section in the context-sensitive task menu on the right.

• Click 5-base methylation QC.

There is no parameters setting required for the 5-base methylation QC task. After click on the 5-base methylation QC task from the context-sensitive task menu, a task node called 5-base methylation QC report is initiated. When completed, double-click on the 5-base methylation QC report task node to open the QC report in a data viewer. The QC report consists of plots and tables organized in 2 sheets. Click on sheet name at the bottom of the data viewer to navigate from one sheet to another.

Metrics

Sheet Metrics shows sample-level QC metrics plot. Each sample is a data point, they are randomly spead out on x-axis. The QC metric is represented by y-axis. Each plot is overlay with a violin plot to show distribution of the QC metrics.

• Percent methylation in samples: Percentages of CpG methylation in samples.

• Percent methylation in unmethylated control: Percentage of CpG methylation in the unmethylated control (lambda). Low value indicates good quality.

• Percent methylation in methylated control: Percentage of CpG methylation in the methylated control (pUC19). High value indicates good quality.

• Percent duplicate reads: Percentage of duplicate marked reads, as a result of PCR amplification.

• Percent mapped reads: Percentage of mapped reads, indicate the alignment rate.

• Average autosomal coverage: Mean autosomal coverage across the whole genome. Higher coverage indicates the counts of methylated/unmethylated more accurately reflects the true methylation amount at any particular site.

• QC metrics table: Text representations of the QC metrics plots.

In Metric sheet, samples can be selected using Selection > Select & Filter. The Select & Filter dialog is pre-loaded with the selection criteria, one for each QC metric.

Hovering the mouse over one of the selection criteria reveals a histogram showing you the frequency distribution of the respective QC metric. The minimum and maximum thresholds can be adjusted by clicking and dragging the sliders or by typing directly into the text boxes for each selection criteria.

Adjusting the selection criteria will select and deselect samples in all 6 plots simultaneously. Depending on your settings, the deselected points will either be dimmed or gray. The filters are additive. Combining multiple filters will include the intersection of the the filters. The number of samples selected is shown in the figure legend of each plot.

To filter the dataset to the selected samples, click the include selected points icon ( ) in Filter in the top right of Select & Filter, and click Apply observation filter...

Select the input data node for the filtering task and click Select.

A new data node, Filtered samples, will be generated under the Analyses tab.

M-bias

Sheet M-bias shows M-bias plots for methylation level and coverage across positions on read1 and read2. The M-bias should be consistent across all positions. It is common for the first/last 10 bases to have un-even methylation due to end-repair and sequencing artifacts.

All plots in one data viewer screen can be downloaded into local computer as a single image by clicking Export button on the top of the screen. To download an individual plot into local computer, select the plot, click Plot button from the left panel within the plot, then click Export, follow the wizard to set image file format, image size, and resolution.