I have sequenced, after bisulfite (CpGenome, Chemicon), first PCR: 45 cycles, second nested PCR 30-35 cycles (SigmaREDTaq) and subsequent cloning, a 635 bp product, containing around 60 CpGs. Sources are different genomic DNA samples.
Although my results are negative for hypermethylation

Is this an artifact? (Due to 2x PCR? it occurs also in a few samples which I cloned without nested PCR) (Due to bisulfite? it doesn't occur in all samples treated at the same time).
A suggested mutation in the genomic DNA does not make sense to me, since normally this cytosine would have been converted after bisulfite (I'm now anyway trying to sequence directly).
I would be grateful to all suggestions.