Hi everyone. I have sequencing data (illumina Hiseq) from bisulfite treated samples (Arabidopsis). The bisulfite conversion of some selected regions was tested by sanger sequencing initially and looked fine. The library was then made from the same samples using the Diagenode microplex kit.
Here's the problem: almost all of the cytosines show either total, or zero methylation levels. There are very few (<1%) that show any intermediate levels of methylation. Even on the regions that were already tested by sanger sequencing, which showed a complete range of methylation levels from 0 to 100%.
So, does anyone know how this bias can come about. My suspicion is that it is either due to an amplification bias when making the libraries, or something in the bioinformatics pipeline. Has anyone experienced similar results from bisulfite sequencing?
Cheers
Here's the problem: almost all of the cytosines show either total, or zero methylation levels. There are very few (<1%) that show any intermediate levels of methylation. Even on the regions that were already tested by sanger sequencing, which showed a complete range of methylation levels from 0 to 100%.
So, does anyone know how this bias can come about. My suspicion is that it is either due to an amplification bias when making the libraries, or something in the bioinformatics pipeline. Has anyone experienced similar results from bisulfite sequencing?
Cheers
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