DNA methylation analysis of the target gene - (Feb/28/2006 )
I am trying to check the methylation state of the target gene (transgene inserted in Nicotiana benthamiana plants) by BSP. For this, I have bisulfite treated the genomic DNA (using Zymo kit) and did PCR using degenerate primers. Then I cloned the PCR product into pGEM-T vector and sequenced using BigDye terminal cycle sequenceing kit. Now I need to analyze the sequence, but confused how to do it. Can anybody help to inform what kind of software do I need to analyze the sequence for methylation? Another problem is that the picks are very week in the sequence chromatogram. Any comments or suggestions will be very nice to me, as I am quite novice in this field.
If you got weak peaks for cloned sequence, the quality of sequencing doesn't sound right to me.
Regarding sequencing result analysis, I would say no software can help much. The best thing you can do is to print out every single sequence using a color printer and analyze them using naked eyes.
I exactly did what you have told me, and noticed interesting results. At the same time I have aligned my original sequence with the bisulfite treated sequence, which also helped me. I am wondering willl I need to prepare the schemetic represrntation of these data manusally or by some software? I often find very nice schemetic diagram of the bisulfite sequencing, but couldn't manage to know how they prepared it. Can anybody help me in this regard?