The NEXTflex™ Bisulfite-Seq Kit uses a similar workflow to other DNA-Seq library preparation kits, but with the addition of a bisulfite conversion step. This conversion takes place following adapter ligation, and consists of denaturing and chemically converting all non-methylated cytosines into uracils. The adapters used for this kit contain methylated cytosines, which will maintain their sequences through bisulfite conversion. Following conversion, DNA is amplified by PCR, which converts the fragments back to double-stranded form and yields thymines in place of post-conversion uracils. Another result of the process is that any guanines that were originally paired with non-methylated cytosines will now be adenines.
This method is known as directional or stranded bisulfite sequencing due to the fact that the forward read will only sequence the Original Top (OT) and Original Bottom (OB) strands that have C → T conversions, and the reverse read (if paired-end sequencing is performed) will only sequence the complement to the OT and OB strands which contain G → A conversions.