Evaluation of bisulfite kits for DNA methylation profiling in terms of DNA fragmentation and DNA recovery using digital PCR

Abstract

DNA methylation is one of the most important epigenetic modifications in the regulation of gene transcription. The current gold standard to study this modification is bisulfite sequencing. Although multiple commercial bisulfite treatment kits provide good conversion efficiencies, DNA loss and especially DNA fragmentation remain troublesome. This hampers DNA methylation profiling of long DNA sequences. Here, we explored the performance of twelve commercial bisulfite kits by an in-depth comparison of DNA fragmentation using gel electrophoresis, qPCR and digital PCR, DNA recovery by spectroscopic measurements and digital PCR and conversion efficiency by next generation sequencing. The results show a clear performance difference between the bisulfite kits, and depending on the specific goal of the study, the most appropriate kit might differ. Moreover, we demonstrated that digital PCR is a valuable method to monitor both DNA fragmentation as well as DNA recovery after bisulfite treatment.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

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.

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.