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

<div><p>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.</p></div

Effect of time and temperature.

<p>Effect of time and temperature of the conversion protocol on fragmentation between the different bisulfite conversion kits. Labels refer to the kit numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199091#pone.0199091.t001" target="_blank">Table 1</a>. 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.</p

Cq values ± SD of the smallest and the largest amplicons.

<p>The data used are the geometric means of the average values from the five donor samples as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199091#pone.0199091.s003" target="_blank">S3 Table</a>. The data labels refer to the kit number as provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199091#pone.0199091.t001" target="_blank">Table 1</a>.</p

Overview over the kits and their main characteristics and performance results.

<p>Overview over the kits and their main characteristics and performance results.</p