Disclosing bias in bisulfite assay: MethPrimers underestimate high DNA methylation

Andrea Fuso, Giampiero Ferraguti, Sigfrido Scarpa, Isidre Ferrer, Marco Lucarelli

Research output: Contribution to journalArticle

Abstract

Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated) versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of "Methylation-Insensitive Primers" (MIPs), having degenerated bases (G/A) to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.

Original languageEnglish
Article numbere0118318
JournalPLoS One
Volume10
Issue number2
DOIs
Publication statusPublished - Feb 18 2015

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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    Fuso, A., Ferraguti, G., Scarpa, S., Ferrer, I., & Lucarelli, M. (2015). Disclosing bias in bisulfite assay: MethPrimers underestimate high DNA methylation. PLoS One, 10(2), [e0118318]. https://doi.org/10.1371/journal.pone.0118318