CpG Methylation Calculator
Calculate CpG observed/expected ratio, methylation percentage, and CpG island identification.
Why CpG Is Depleted from the Genome
CpG dinucleotides occur at roughly a fifth of the expected frequency across mammalian genomes, and the reason is chemical. Methylated cytosine deaminates spontaneously to thymine — a change that produces a normal T:G mismatch which repair machinery often resolves the wrong way.
Unmethylated cytosine deaminates to uracil instead, which is immediately recognised as foreign in DNA and excised. So methylation converts a repairable lesion into a permanent mutation, and over evolutionary time CpG sites have been progressively lost.
| O/E ratio | Interpretation |
|---|---|
| ~0.2 | Genome average — CpG depleted |
| > 0.6 | CpG island — historically unmethylated |
| > 0.65 with GC > 55% | Strict island definition |
CpG islands survive precisely because they are not methylated in the germline, so they escape the deamination pressure. Their existence is evidence of their own unmethylated history.
Methylation and Cancer
| Region | Normal tissue | Typical tumour |
|---|---|---|
| Promoter CpG islands | Unmethylated | Hypermethylated — tumour suppressors silenced |
| Genome-wide | Methylated | Hypomethylated — instability, transposon activation |
The two changes occur together and point in opposite directions, which is why describing cancer as "hypermethylated" or "hypomethylated" is incomplete. Focal promoter hypermethylation silences genes such as MLH1 and CDKN2A, while global hypomethylation destabilises the genome.
This matters therapeutically: hypomethylating agents such as azacitidine aim to reverse the promoter silencing and restore tumour suppressor expression.
Measuring It
Bisulfite conversion deaminates unmethylated cytosine to uracil while leaving 5-methylcytosine intact, so methylation becomes a sequence difference. One caveat: standard bisulfite sequencing cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine, since both resist conversion. Oxidative bisulfite is required to separate them.
Worked Examples
Common Mistakes
Tumours typically show focal promoter hypermethylation alongside global hypomethylation. The two occur together and have opposite consequences.
Both resist bisulfite conversion, so standard sequencing cannot distinguish them. Oxidative bisulfite is needed to separate the two marks.
Standard definitions also require GC content above 50–55% and a minimum length, typically 200 bp or more.
The relationship is strong for promoter islands but gene body methylation often correlates with active transcription instead.