Partial methylation/incomplete modification – BSP - (Oct/12/2005 )
I'm performing bisulfite sequencing on tumour cell lines as well as healthy controls.
In one of my genes, all non-CpG C's are converted to T's. At the sites of the CpG C's I have both T's and C's, indicative of partial methylation... Or could this be due to incomplete conversion at these specific sites??? Would you say that I have methylation or partial methylation?
Thanks in advance!
I assume you are performing direct sequencing? if so then what you are seeing is indeed partial methylation, if you were sequencing clones then I would say you have a mixed colony.
All non CpG C's converting to T is a good sign that you are dealing with a fully converted sequence
Yes, it's direct sequencing. I have a feeling that the degree of partial methylation differs some between modifications on the samples, the ration between the C and T peak is a bit diffrent from time to time although all non-CpG C's are fully converted. At one run I hardly have any T peak and in the next the C and T could be of almost equal size. Do you have an explaination for this?
I can think of two reasons,
1. you have a mixed population of cells that contain in some, the CpG island is methylated while in others it is not. The the proportion of mixed cells is significant if you can see both methylated and unmethylated forms in a direct sequencing run.
2. the cpg island you are looking at is imprinted.
unfortunately there is not a way of distinguishing between the two unless you already know that your gene is indeed imprinted.
There is a good survey of imprinted genes in human by Morison et al in trends in genetics if I recall correctly.