De-novo CpGs

In addition to looking at methylation of known CpG sites, i.e. sites that are already present in the reference genome, Rastair can also call de-novo CpG sites. These are sites where a C and/or G alternative allele exists in reads followed/preceded by a G and/or C reference allele, and thus creating new CpG sites.

Example

Here is an example of a de-novo CpG site in a pileup:

Position    1 2 3 4 5 6 7 8

Reference:  A T C C T A G C  Strand

Reads:      A T T G T A G C    +
            A T C A T A G C    -
            A T C A T A G C    -
            A T T G T A G C    +
            A T T G T A G C    +
                  ↑
                  De-novo CpG created by C>G variant

In this example, some reads have a G at position 3 where the reference has a C. This creates a new CpG dinucleotide (CG) that is not present in the reference genome. This newly generated CpG is methylated, which means that both the C and the G position will show T/A at OT/OB reads, respectively!

Methylation of de-novo CpGs

After Rastair has identified de-novo CpG sites, it will also call methylation for these sites. For a methylated de-novo CpG site, there have to be both C and T (or G and A) alternative alleles, which means the amount of evidence present is generally lower than for known CpG sites.

The same filter criteria as for known CpG sites are applied.