incomplete conversion - how do you tell? - (Aug/02/2005 )
Wow! You guys are good!
Please help me out here.
After BS sequencing, I notice that on the chromatogram, the C and the T peaks are quite similar -- yet the sequencing facility that I use gives me a T in most cases. Is this because there is incomplete conversion at that particular nucleotide?? Do mCs get converted as well? Under what circumstances??
I am dealing with viral DNA, so it might not necessarily be CpG that I am looking at.
I don't know viral DNA methylation. For DNA from mammalian cells, if it is not fully converted by bisulfite, you will notice "C"s at non-cpg 'C' on the chromatogram. Although there are a few papers reporting non-cpg C methylation in human cells, it is still a controversial issue; if DNA is over converted (for example, longer reaction time) some 5mCs will show up as T, which is harder to tell than incomplete conversion because you don't know if the CpG site is truly unmethylated or over converted. So the conversion reaction should not last for more than 16 hrs.
With the basecalling, I am aware that ABI's basecalling will compensate for the defiecent C signal within the chromatogram, so you may well be seeing just the background.
I am not too sure if there is a way to get around this, but if you check the fluorescence signals you will find that the C signal would be a lot lower than the other 3 channels therefore you may get what you are seeing.
Thanks so much for the prompt replies.
Some C >T at CpG sites!
During conversion with bisuphite, I carried out the incubation for 16h.
I'll probably send more samples for sequencing then...
please help me out here.
Suppose I transform my target DNA (after BS conversion) into E Coli after TA cloning. How many clones do I send for sequencing? Is there a standard?
How accurate is direct sequencing?
I am getting increasingly frustrated becos out of the 6 samples i send for direct sequencing, 3 showed 'T' and 3 showed 'C'! And this is not a CpG. It's CpT. How do I resolve this? Is this a true result?
Thanks so much!