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:

Reference:  T C C T A  Strand
Reads:      . T G . .    +
            . . A . .    -
            . . A . .    -
            . T G . .    +
            . T G . .    +
                ↑
                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.

The same pairwise logic described here for regular methylation is applied: Only CG, TG, and CA pairs are considered.