Call Methylation Levels

The tool takes as input one or more read mappings created by the Map Bisulfite Reads to Reference tool. If more than one mapping is used as input, various statistical options to detect differential methylation become available.

The tool will accept a regular mapping as input, but will warn about possibly inconsistent interpretation of results. Mapping done in a 'normal', not bisulfite mode, is likely to result in sub-optimal placement of reads due to a large number of C/T mismatches between bisulfite-converted reads and a reference. Also, this tool will consequently interpret majority of cytosines in a reference as methylated, creating possibly very large and misleading output files. The invisible 'bisulfite' property of a mapping may be erased if the original mapping is manipulated in the workbench with other tools - such as the Merge Read Mappings tool - in which case the warning should be ignored.

After selecting the relevant mapping(s), the wizard offers to set the parameters for base-level methylation calling, as shown in figure 36.25:

Image methylation-call-settings
Figure 36.25: Methylation call settings.

Statistical tests and thresholds settings

The next set of parameters depends on experimental setup, and a number of samples in the input, as shown in 36.26.

Image statistical-tests
Figure 36.26: The statistical tests and thresholds settings.

Statistical test

Window thresholds

Sample thresholds

The tool produces a number of feature tracks and reports. Select the outputs you are interested in during the last wizard step of the tool. The Create track of methylated cytosines option is chosen by default. It will provide a base level methylation track for each read mapping supplied, i.e., case or control (see figure 36.27 for a table view of the track).

Image bs-tableview-1
Figure 36.27: Output table.

In the table, each row corresponds to a cytosine that conforms to a context (such as 'CpG' in this example) and which has non-zero methylated coverage.

The columns of the methylation levels track table view indicate:

For each mapping, you can also generate an optional summary report by selecting the Create methylation reports option. This report includes statistics of direction of mapping of reads/read pairs, chosen contexts, and useful graphs. The graphs can help detect any bias in called methylation levels that commonly occurs at the start of BS-seq reads due to end-repair of DNA fragments in library preparation. This facilitates setting the correct trimming parameters for Read 1 soft clip, Read 2 soft clip.

Note that positions where no methylation was detected are filtered from the final output and are not reported in the 'Methylation levels' feature track. However they are included in the intermediate calculations for differential methylation detection.

When the statistical test is performed, a feature track is produced. If more than one methylation context is chosen, a separate feature track is produced for each individual context, i.e., for CpG, CHH, etc. The table view of such track for Fisher exact test is shown in figure 36.28.

Image bs-tableview-2
Figure 36.28: Example of table with statistical test output.

The columns of the differential methylation feature track table indicate:

For the highlighted window region 833001..834000, the relevant values used in the hypergeometric test are 6 (the number of methylated cytosines in the case sample) out of 7 (total number of cytosines), while the control sample had 11 covered context-conforming cytosines in the region, of which only 2 were methylated. If there are no case/control difference in methylation, the probability (p-value) of such hypermethylation in the case sample is calculated as $ 9.05\times10^{-3}$, below the threshold.