Pyrosequencing Evaluation of Widely Available Bisulfite Conversion Methods: Considerations for Application

DSpace/Manakin Repository

Pyrosequencing Evaluation of Widely Available Bisulfite Conversion Methods: Considerations for Application

Citable link to this page

 

 
Title: Pyrosequencing Evaluation of Widely Available Bisulfite Conversion Methods: Considerations for Application
Author: Izzi, Benedetta; Binder, Alexandra M.; Michels, Karin B.

Note: Order does not necessarily reflect citation order of authors.

Citation: Izzi, Benedetta, Alexandra M. Binder, and Karin B. Michels. 2014. “Pyrosequencing Evaluation of Widely Available Bisulfite Conversion Methods: Considerations for Application.” Medical epigenetics 2 (1): 28-36. doi:10.1159/000358882. http://dx.doi.org/10.1159/000358882.
Full Text & Related Files:
Abstract: Introduction: 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 of ALUSX, IGF2 and ST3GAL2, while the conversion of the low methylated IL6ST 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 IL6; 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.
Published Version: doi:10.1159/000358882
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4058339/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:13890620
Downloads of this work:

Show full Dublin Core record

This item appears in the following Collection(s)

 
 

Search DASH


Advanced Search
 
 

Submitters