Date Published: June 14, 2018
Publisher: Public Library of Science
Author(s): Sam Kint, Ward De Spiegelaere, Jonas De Kesel, Linos Vandekerckhove, Wim Van Criekinge, Emidio Albertini.
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.
DNA methylation (5-methylcytosine) is an epigenetic modification that is typically associated with stable transcriptional silencing [1–4]. This modification plays an important role in several biological processes associated with development and disease. Examples are cell differentiation, regulation of gene expression, X-chromosome inactivation and genomic imprinting [1,5,6]. In disease, DNA methylation is heavily involved in the development of genetic diseases, carcinogenesis and silencing of intracellular viruses [7–17]. Consequently, DNA methylation provides a promising diagnostic tool in medicine. Previous studies to understand the exact role of DNA methylation in these disease settings have already resulted in several clinically validated biomarkers (e.g. MGMT promoter methylation in patients with glioblastoma (PredictMDx, MDxHealth, Inc.); methylation of GSTP1, APC and RASSF1 genes for prostate cancer testing (ConfirmMDx, MDxHealth, Inc.); methylation of PITX2 in Formalin-Fixed, Paraffin-Embedded prostatectomy specimens for identifying patients who are at high risk to suffer from prostate-specific antigen recurrence after radical prostatectomy; free-circulating methylated SEPT9 gene copies in plasma as a screening biomarker for colorectal cancer; SHOX2 DNA methylation as a plasma based biomarker for detection of lung cancer) [18–28].
The importance of DNA methylation in different biological processes and the need for an easy-to-use technology have resulted in a wide range of commercially available bisulfite conversion kits. To select the most appropriate bisulfite conversion kit for specific applications, several parameters have to be taken into account. In the current study, we provide a comprehensive workflow for analyzing bisulfite kit performance by comparing twelve bisulfite kits based on DNA recovery and DNA fragmentation. Since dPCR provides a direct absolute quantification with minimal influence of variations in PCR efficiency , we can use one method to directly compare samples before and after bisulfite conversion, enabling us to investigate both DNA recovery and fragmentation with more ease and higher accuracy compared to classical methods. The present work shows the strength of dPCR for analysis of the quality of bisulfite treated DNA.