title: Pyrosequencing Evaluation of Widely Available Bisulfite Conversion Methods: Considerations for Application.
abstract: BACKGROUND
  Bisulfite treatment of DNA introduces methylation-dependent sequence changes through selective chemical conversion of nonmethylated cytosine to uracil and serves as pretreatment step for the majority of DNA methylation analysis methods.
  
  
  METHODS
  We have evaluated the conversion performance of five of the most commonly used bisulfite treatment kits [MethylDetector (Active Motif), Epitect+ (Qiagen), Zymo Methylation, Zymo Gold and Zymo Lightning (all from Zymo Research)] by pyrosequencing four different regions with variable methylation levels, including: a repetitive element (ALUSX), a gene with low levels of methylation (IL6ST), an imprinted gene expected to be approximately 50% methylated (IGF2), and a fully methylated gene (ST3GAL2). In addition, we have studied the influence of duration (3 vs. 16 h) and type (fixed temperature vs. cycling program) of incubation protocol on the conversion efficiency of each evaluated kit.
  
  
  RESULTS
  All kits produced similar conversion rates ofALUSX,IGF2andST3GAL2, while the conversion of the low methylatedIL6STgene was variable between kits. The Zymo kits were highly consistent in their performance even when different protocols of incubation were applied, generating full conversion at the low methylated geneIL6; this was not true for the MethylDetector and Epitect+ kits. However, long-cycling incubation could produce similar conversion rates for the same locus in combination with Active Motif and Qiagen kits.
  
  
  CONCLUSIONS
  The selection of a long-cycling protocol during conversion permits standardization of protocols, improving the reproducibility of methylation estimates across laboratories for gene-specific, genome-wide and bisulfite-based sequencing analyses.
authors.affiliation: Institute for Prevention and Cancer Epidemiology, Freiburg Medical Center, University of Freiburg, Freiburg, Germany ; Obstetrics and Gynecology Epidemiology Center, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass., USA.
authors.forename: Benedetta
authors.initials: B
authors.lastname: Izzi
authors.affiliation: Harvard School of Public Health, Department of Epidemiology, Boston, Mass., USA.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Binder
authors.affiliation: Institute for Prevention and Cancer Epidemiology, Freiburg Medical Center, University of Freiburg, Freiburg, Germany ; Obstetrics and Gynecology Epidemiology Center, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass., USA ; Harvard School of Public Health, Department of Epidemiology, Boston, Mass., USA.
authors.forename: Karin B
authors.initials: KB
authors.lastname: Michels
keywords: Bisulfite treatment; DNA methylation; Pyrosequencing Abstract