Date Published: August 6, 2015
Publisher: Public Library of Science
Author(s): Chrysanthia A. Leontiou, Michael D. Hadjidaniel, Petros Mina, Pavlos Antoniou, Marios Ioannides, Philippos C. Patsalis, Osman El-Maarri.
Epigenetic 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.
Performance 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.
The 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.
Our 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.
Prenatal testing is a significant part of modern obstetric care and its primary aim is the diagnosis of fetal genetic abnormalities . In Europe, the prevalence of chromosomal abnormalities for all pregnancies is 3.6 per 1,000 births . Trisomies 21, 18 and 13 and sex chromosome anomalies are the most common identified among the live births with aneuploidies . Currently, the main goal of prenatal testing is to provide parents with the choice to abort a fetus with the diagnosed condition or to prepare psychologically, socially, financially and medically for a child with a health problem or disability, or for the likelihood of a stillbirth . The most common methods used for prenatal diagnosis are chorionic villi sampling (CVS) during the first trimester and amniocentesis during the second trimester, with their diagnostic accuracy estimated to be 98 to 99% [5, 6]. Both of these procedures are invasive with a significant risk of fetal loss, between 0.5 to 1% of all tested cases [7–9]. Therefore, these invasive tests are currently performed only in high-risk pregnancies or in pregnancies with increased maternal age and/or family history of having a child with an inherited disease.
DNA methylation analysis has undergone a major technological revolution in recent years. Especially, bisulfite conversion when coupled to NGS, has enabled genome wide methylation analysis in a high throughput manner at single base resolution with a quick turn-around time. Aggressive bisulfite treatment protocols (long incubation, high temperatures, high molarity of bisulfite) secure full conversion of cytosines to uracils, but DNA can be degraded to a degree that makes PCR amplification impossible. On the other hand, less aggressive treatments bear the risk of incomplete conversion and therefore can lead to overestimation of the methylation levels. Therefore, the challenge of the method is that the desired outcomes (conversion of unmethylated cytosines) positively correlate with the undesired side effects (DNA degradation and inappropriate conversion) and one should aim to find the best possible balance between the two. Several kits are commercially available for bisulfite conversion of DNA, each with their own advantages and disadvantages. One of the most important parameters of bisulfite conversion is the yield of DNA post treatment. It is crucial to be sufficiently high for downstream molecular analyses. Furthermore, the incomplete and inappropriate conversion during bisulfite treatment is another imperative factor and should be considered carefully. In this study we aimed to compare four different bisulfite conversion kits with regards to their conversion efficiency, DNA degradation and conversion specificity. The kits that were compared were the Premium Bisulfite kit (Cat. No. C02030030, Diagenode), the EpiTect Bisulfite kit (Cat. No. 59104, Qiagen), the MethylEdge Bisulfite Conversion System (Cat. No. N1301, Promega) and the BisulFlash DNA Modification kit (Cat. No. 1026, Epigentek).
The kits studied in this project showed similar yet distinct results regarding DNA degradation, conversion efficiency and conversion specificity. For the purposes of our experiments, in our laboratoty and based on the results that are presented in this study, it was concluded that the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). Furthermore, two of our previously published DMRs were studied for the first time at single base resolution and their methylation difference between CVS and whole blood was confirmed. Finally, we have shown that bisulfite amplicon sequencing is a suitable approach for methylation analysis of specific regions.