Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

Abstract

IntroductionEpigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity.MethodsPerformance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood.ResultsThe kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood.ConclusionOur findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.

METHimpute: Imputation-guided construction of complete methylomes from WGBS data

“…Some controls (commercial or artificially methylated DNA fragments) are available to estimate this inappropriate conversion rate, but, to our knowledge, they are not systematically used in WGBS experiments. Some studies using such controls have shown that the inappropriate conversion rate (% of methylated cytosines converted to uracils) ranges from 0.09% to 6.1% depending on the kit and protocol used [56][57][58].…”

An Internal Control for Evaluating Bisulfite Conversion in the Analysis of Short Stature Homeobox 2 Methylation in Lung Cancer

“…However, the low pH and high temperature in this reaction can lead to incomplete or inappropriate bisulfite conversion as well as DNA degradation, resulting in over or underestimation of DNA methylation (Olova et al, 2018;Ji et al, 2014;Genereux et al, 2008). Recently, different commercial bisulfite conversion kits, applied to various DNA sources with high and low molecular weigh (HMW/LMW), have been comprehensively evaluated for their DNA recovery and bisulfite conversion efficiencies (Holmes et al, 2014;Leontiou et al, 2015;Ørntoft et al, 2017;Tierling et al, 2018). In these performance evaluations, highly sensitive detection methods such as quantitative and digital PCR (qPCR, dPCR) were used to amplify different target regions in genomic DNA (HMW-DNA) or in circulating cell-free DNA (LMW-DNA) (Ørntoft et al, 2017;Tierling et al, 2018;Kint et al, 2018).…”

Population genomic SNPs from epigenetic RADs: gaining genetic and epigenetic data from a single established next-generation sequencing approach

“…One example is bisulfite sequencing, which comes in a number of variations (whole genome, reduced representation, target sequencing of specific gene regions) and has been shown to provide high resolution of the methylation landscape within genomes (Metzger & Schulte, 2016). However, this technique is expensive, can result in excessive DNA degradation, and requires a related reference genome for the species of interest, something that is still lacking for most non-model organisms (Leontiou et al 2015; Metzger & Schulte, 2016).…”