Hello,
I am a immunology researcher from Japan.
I am performing epigenetic analysis of several lymphocytes.
There are regions whose methylation status are related to lineage commitment and confirmed by bisulfite sequence.
Problem is that regions that were demethylated (confirmed by bisulfite sequencing) showed high peak in MBD-seq results. The feature of this region is high CpG density and 200bp length.
I used Methylmeiner from invitrogen for MBD-seq.
The fragment sizes is about 100-120bp and read by Ion Proton.
Most of regions of MBD-seq results showed similar results as Bisulfite-seq.
But only one region showed discrepancy between two methods.
And more only one sample showed this discrepancy, other sample didn't show this, which means samples whose same region are methyalated showed high peak in MBD and samples whose same region are demethylated showed no peak.
So, it is not the problem of mapping or other analysis method.
This discrepancy is biological duplicated.
What do you think about this discrepancy?
Are there any critical problem on MBD-seq or Bisulfite-seq.
For example
1. Primers for bisulfite-seq is designed on the basis that non CpG is demethylated.
2. Too much MBD against DNA or too much DNA against MBD lead to such discrepancy.
.....
If MBD-seq is not reliable one, I cannot use this method.
Best,
gatapishi
I am a immunology researcher from Japan.
I am performing epigenetic analysis of several lymphocytes.
There are regions whose methylation status are related to lineage commitment and confirmed by bisulfite sequence.
Problem is that regions that were demethylated (confirmed by bisulfite sequencing) showed high peak in MBD-seq results. The feature of this region is high CpG density and 200bp length.
I used Methylmeiner from invitrogen for MBD-seq.
The fragment sizes is about 100-120bp and read by Ion Proton.
Most of regions of MBD-seq results showed similar results as Bisulfite-seq.
But only one region showed discrepancy between two methods.
And more only one sample showed this discrepancy, other sample didn't show this, which means samples whose same region are methyalated showed high peak in MBD and samples whose same region are demethylated showed no peak.
So, it is not the problem of mapping or other analysis method.
This discrepancy is biological duplicated.
What do you think about this discrepancy?
Are there any critical problem on MBD-seq or Bisulfite-seq.
For example
1. Primers for bisulfite-seq is designed on the basis that non CpG is demethylated.
2. Too much MBD against DNA or too much DNA against MBD lead to such discrepancy.
.....
If MBD-seq is not reliable one, I cannot use this method.
Best,
gatapishi