Hello,
we had a problem during our Pyrosequencing (at Pyro Q24 analyser).
Some of them are:
1) Methylation CTRL at 30% (instead of 100%) at all 5 CpG sites
2) unmetylated CTRL giving identical results, with blank sample clean (no contamination)
3) peak intensity is higher than it should be for first part of the sequence, but put in relation to other referece nucleotide signals, software was able to read the sequence correctly
What might be the issue?
I would say it could be conversion efficiency or low sequencing primer efficiency.
Thank you for your answer. We have one C outside CpG (bisulphite conversion control point) which is marked by PyroMarko as “inner control of conversion” and it gives us result of full conversion.
All similar results might be due to systemic contamination (probably mastermix). We will try to run PCR with new kit.
Another thing is: DNA was isolated from FFPE slices of cancerous tissue, and pathologists gave astimation on %of tumor cells. I expect higher methylation in cancer cells and
I don't know the reason for the first two problems.
For #3:
With Sanger Sequencing, you see that pattern when the primer concentration is too high. The signal will be too high at the beginning of the sequence. The signal drops off quickly and is too low after 100 - 200 bp. Could you be having the same issue with Pyrosequencing?