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.

01. Fragmentation

Sample DNA is fragmented into smaller length pieces containing jagged ends.

Bisulfite-Seq Fragmentation
Bisulfite-Seq End Repair & Adenylation

02. End Repair & Adenylation

5’ overhangs are filled in and 3' overhangs are cut back. 5’ ends are phosphorylated.
3’ end is then A-tailed in a separate step after end repair.

03. Ligation

“Y” shaped adapters are added to both ends of the end repaired/adenylated fragments.

Bisulfite-Seq Ligation
Bisulfite-Seq Bisulfite Conversion

04. Bisulfite Conversion

DNA is denatured and unmethylated cytosines are converted to uracils.

05. PCR Cycle I

Original Top (OT) (blue) and Original Bottom (OB) (orange) strands are used as template. Uracils pair with adenines, therefore guanines that had been paired with unmethylated cytosines will now be adenines.
The binding site for primer two is created through extension of the Y strand.

Bisulfite-Seq PCR Cycle 1

06. PCR Cycle II

Complements to OT and OB strand are used as template for primer 2. Uracils are replaced with thymines in original strands,
therefore unmethylated cytosines have gone from C → U → T.

Bisulfite-Seq PCR Cycle 2

07. Post-PCR Products

Post-PCR products consist of four possible strands. OT in blue, OB in orange, Complement to Original Top (CTOT) in purple, and Complement to Original Bottom (CTOB) in maroon.
Cytosines are no longer methylated since they are PCR products, but they are still labeled as such in the diagram as a reminder that they were not converted.

Bisulfite-Seq Post-PCR Products

08. Sequencing Read 1: C → T

This read will only sequence the OT and OB strands. All conversions in the read will be C → T.
Bases added during end repair step will only be sequenced at the end of the read if the read length is longer than the insert.
A common strategy in bisulfite data analysis is to trim back 3-6 bases on the 5’ end after the adapter is trimmed for R1.

Bisulfite-Seq Sequencing C → T

09. Sequencing Read 2: G → A

This read will only take place in paired-end sequencing, and will sequence the CTOT and CTOB strands.
All conversions will be G → A and bases added during the end repair step will be read at beginning of the read.
A common strategy for data analysis is to trim the first 3-6 bases off of the beginning of R2.

Bisulfite-Seq Sequencing G → A