We are going to experiment on rats (Developer version of this kit). We want to use this kit to determine the methylation status at a defined group of genes.
How much DNA can be determined from a single reaction (pooled samples)?
My next question concerns the coverage. We have mothers and their children, on various diets. What coverage is essential for proper bioinformatical evaluation? (we have Illumina sequencer)
Can anyone please help me I will be grateful.
Hi Josef,
On the second aspect of your question, the required coverage is dependent upon the methylation change (Delta-beta) that you expect to see. For example, in cancer it is common for completely unmethylated sites to become fully methylated, so in 80% pure solid tumour, we expect 8 of 10 reads to be methylated and in matched normal tissue 0 of 10 (0%) of reads to be methylated. With more subtle phenotypes under inspection, the coverage must be far greater.
Given a particular minimal delta beta you wish to find, say 0.1, a rudimentary Poisson statistic-based power analysis can be used to find what number of reps and coverage is required. Of course this doesn't account for multiple comparisons, but then DMR finders can help here (such as DMRcate or Bumphunter).
Hi Josef,
As I know, the most sensitive method is a PCR based one, which can quantitate gDNA global methylation percentage in single cell level and also can be analyzed with Illumina sequencer.
good luck
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