<b><i>Introduction:</i></b> 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. <b><i>Methods:</i></b> 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 <i>(ALUSX)</i>, a gene with low levels of methylation <i>(IL6ST)</i>, an imprinted gene expected to be approximately 50% methylated <i>(IGF2)</i>, and a fully methylated gene <i>(ST3GAL2)</i>. 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. <b><i>Results:</i></b> All kits produced similar conversion rates of <i>ALUSX,</i><i>IGF2</i> and <i>ST3GAL2</i>, while the conversion of the low methylated <i>IL6ST</i> gene 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 gene <i>IL6</i>; 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. <b><i>Conclusions:</i></b> 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
