Validating Methylation Analysis with Pyrosequencing

Infinium HumanMethylation450 BeadChip is a current and reliable array to detect CpG methylation.^{15 (link)} However, we propose to compare some of our findings using a technical reference based on pyrosequencing.^{16 (link)} After bisulfite conversion by EpiTect Plus Bisulfite Kits (Qiagen, Hilden, Germany) and DNA purification on column, non-methylation specific PCR were achieved using Platinium Taq DNA polymerase kit (Invitrogen—Life Technologies, Carlsbad, CA, USA). MAEL promoter was used as positive control for the bisulfite treatment^{17 (link)} in bisulfited and non-bisulfited samples (Supplementary Figures S4 and S5). Two findings were assessed: we chosen to confirm one specific CpG selected from the top results of multi-CpGs analysis (CpG located in CHL1 gene) and one significant DMR in GSTM5 (the only one significant DMR including a promoter) identified by the Minfi package. Primers were designed by the PyroMark Assay design Software 2.0 (Qiagen), and technical conditions for PCR are shown in Supplementary Table S3; examples of results are shown in Supplementary Figure S6. Biotinylated primers were used to keep the single DNA strand for pyrosequencing. Pyrosequencing was performed using PyroMark Q24 (Qiagen) according the manufacturer's instructions, and data about methylation in each CpG were extracted and analyzed using the PyroMark Q24 2.0.6.20 software (Qiagen).