Fig 1.
Principle of bisulfite-mediated methylcytosine (mC) mapping.
A: Deamination of cytosine (C) and mC. Sodium bisulfite deaminates C to uracil (U) (upper row) and mC to thymine (T) (lower row). The rate of mC deamination is two orders of magnitude less than that of C. B: The mapping protocol: after bisulfite treatment, mC will remain C where unmethylated C will be deaminated to U. During subsequent PCR amplification, the U deamination product templates adenine (A), which then templates T, resulting in a C to T transition at unmethylated C. By sequencing, mC can be identified as bases that remained C after bisulfite treatment [38].
Fig 2.
DNA recovery of the twelve bisulfite kits.
DNA recovery is shown as percentages ± SD and is calculated by the ratio of the measured output concentration of each bisulfite kit (Qubit ssDNA) to the maximal theoretical output concentration based on the input in every kit (Qubit dsDNA). The labels in the figure refer to the kit numbers in Table 1.
Table 1.
Overview over the kits and their main characteristics and performance results.
Fig 3.
Cq values ± SD of the smallest and the largest amplicons.
The data used are the geometric means of the average values from the five donor samples as shown in S3 Table. The data labels refer to the kit number as provided in Table 1.
Fig 4.
Visual representation of the rankings of the dPCR experiments.
Results are given as the geometric means of the average number of intact copies per ng bisulfite treated (BT) DNA measured by dPCR ± SD of all the five donor samples as shown in S4 Table. The data labels refer to the kit number as provided in Table 1.
Fig 5.
Effect of time and temperature.
Effect of time and temperature of the conversion protocol on fragmentation between the different bisulfite conversion kits. Labels refer to the kit numbers in Table 1. The upper panels (A-B) show the effect of different conversion temperatures between the kits measured by qPCR (A) and dPCR (B). The lower panels (C-D) show the effect of different conversion times between the kits measured by qPCR (C) and dPCR (D). The values on the x-axis are normalized to show the relative amount of fragmentation: 0 is least fragmenting and 1 is most fragmenting.