title: 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: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Translational Genetics Team, The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus.
authors.forename: Chrysanthia A
authors.initials: CA
authors.lastname: Leontiou
authors.affiliation: Translational Genetics Team, The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Hadjidaniel
authors.affiliation: Translational Genetics Team, The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus.
authors.forename: Petros
authors.initials: P
authors.lastname: Mina
authors.affiliation: NIPD Genetics Ltd, Nicosia, Cyprus.
authors.forename: Pavlos
authors.initials: P
authors.lastname: Antoniou
authors.affiliation: Translational Genetics Team, The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus.
authors.forename: Marios
authors.initials: M
authors.lastname: Ioannides
authors.affiliation: Translational Genetics Team, The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus; The Ministry of Health, Nicosia, Cyprus.
authors.forename: Philippos C
authors.initials: PC
authors.lastname: Patsalis
mesh_terms: D001483:Base Sequence; D002824:Chorionic Villi; D018899:CpG Islands; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D011296:Prenatal Diagnosis; D011933:Reagent Kits, Diagnostic / Q000592:standards; D013447:Sulfites / Q000737:chemistry*
chemical_list: D011933:Reagent Kits, Diagnostic; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite