TAB-seq βGT protection efficiency note
Please note the following regarding our TAB-seq Kits:
1. 5hmC spike-in control we provide is generated by PCR amplification using 5-hydroxymethylcytosine dNTP mix. In principle all cytosines in 5hmC spike-in control should be 100% hydroxymethylated. However, due to the impurity of commercial 5hmdCTP and the slow oxidation of 5hmC upon exposure to air, the actual abundance of 5hmC at each cytosine site in the 5hmC spike-in control is roughly 80%. As a result, only 80% of cytosines in 5hmC spike-in control will be read as ‘C’ even in standard bisulfite sequencing. This number (~80%) is subject to change depending on the storage condition/time for the 5hmC spike-in control.
To accurately evaluate the protection rate of 5hmC in TAB-seq, we recommend running standard bisulfite sequencing for one sample containing the same batch of 5hmC spike-in control in parallel to determine its real 5hmC abundance. For example, if 80% of all cytosines in 5hmC spike-in control are read as ‘C’ in standard bisulfite sequencing and 70% of them are read as ‘C’ in TAB-seq, then the real protection rate for your TAB-seq assay should be 0.7/0.8=87.5% but not 70%.
2. After βGT protection, the dense 5gmC on the 5hmC control may stall polymerization when certain polymerase is used (e.g. Taq polymerase). To avoid the possible PCR bias, PfuTurbo Cx hotstart polymerase (Agilent) or equivalent is recommended for the final PCR amplification step in library construction.