I have a question regarding the baseline noise for bisulfite sequencing on the ABI3130.
What would you guys think as the acceptable baseline noise for reliable quantification of the methylation percentage at CpGs.
In order to access the completion of my bisulfite conversion, I would need to observe complete C to T conversion at non-CpG regions. However, I am also observing the baseline noise on the sequencing trace of 3%-10%. Is there an acceptable cut off value for baseline noise when assessing bisulfite sequences? I would think it is unrealistic to expect 0% baseline noise...?
Also, if my baseline noise regularly ranges from 3-10%, that would also mean I will never be able to reliably detect methylation levels that are less than 10%, am I right?
Also, I also having problems obtaining reproducible methylation percentage on both the Forward and reverse sequence. Does anyone know why this happens?
Any suggestion and help is deeply appreciated.