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Accurate measurement of 5-methylcytosine and 5-hydroxymethylcytosine in human cerebellum DNA by oxidative bisulfite on an array (OxBS-array).


Type

Article

Change log

Authors

Field, Sarah F 
Beraldi, Dario 
Bachman, Martin 
Stewart, Sabrina K 
Beck, Stephan 

Abstract

The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The "methylation" level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3%. We performed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3% and 42%. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.

Description

Keywords

5-Methylcytosine, Adult, Cerebellum, Cytosine, DNA, DNA Methylation, Humans, Male, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, Reproducibility of Results, Sulfites

Journal Title

PLoS One

Conference Name

Journal ISSN

1932-6203
1932-6203

Volume Title

10

Publisher

Public Library of Science (PLoS)
Sponsorship
Wellcome Trust (099232/Z/12/Z)
S. Balasubramanian is a Senior Investigator of The Wellcome Trust and the Balasubramanian group is core-funded by Cancer Research UK. We would like to thank Tobias Ost and Christine Clark of Cambridge Epigenetix Ltd. for valuable discussions and development of the method.