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

PLoS One. 2018 Jun 14;13(6):e0199091. doi: 10.1371/journal.pone.0199091. eCollection 2018.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / genetics*
  • DNA Fragmentation / drug effects*
  • DNA Methylation / drug effects*
  • Polymerase Chain Reaction / methods*
  • Sulfites / pharmacology*
  • Temperature
  • Time Factors

Substances

  • Sulfites
  • DNA
  • hydrogen sulfite

Grants and funding

Linos Vandekerckhove is funded by the Research Foundation Flanders (www.fwo.be, Grant number 1802019N). Ward De Spiegelaere was a post-doctoral fellow at the Research Foundation Flanders (www.fwo.be, Grant number 12G9716N. Additionally, this work was supported by HIVERA:EURECA, ERANET 01KI1307A.