Figure 5

Pre-sequencing quality control of bisulfite-converted DNA libraries. A) Cloning and subsequent EcoRI release of library inserts reveals a random size distribution as revealed on an agarose gel. The extra band in some lanes represents enzyme cut sites present in the inserts. B) qPCR dissociation curve of a bisulfite- converted DNA library using primers directed at adaptors to the bisulfite library (blue peak) and PhiX standard DNA library (green peak). Bisulfite converted DNA libraries have a lower melting temperature due to loss of cytosine residues. The qPCR dissociation curve only serves as a general tool to verify bisufite conversion and is not able to distinguish a minor bisulfite conversion problem. C) Sanger sequencing reaction of a single clone insert from A). Plasmid is detectable by presence of cytosine residues; insert begins at CGG residue marked by the arrow. Note the lack of cytosine residues in the insert, except at CpG loci (blue ‘C’ peaks under black arrow). Base colors are: C: Blue, G: Black, T: Red, A: Green.