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BMC Biol. 2013 Jan 22;11:4. doi: 10.1186/1741-7007-11-4.

Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation.

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1
Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA.

Abstract

BACKGROUND:

DNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection.

RESULTS:

We characterized the kinetic signatures of various cytosine modifications, demonstrating that 5-carboxylcytosine has a larger impact on the local polymerase rate than 5-methylcytosine. Using Tet1-mediated conversion, we show improved detection of 5-methylcytosine using in vitro methylated templates and apply the method to the characterization of 5-methylcytosine sites in the genomes of Escherichia coli MG1655 and Bacillus halodurans C-125.

CONCLUSIONS:

We have developed a method for the enhancement of directly detecting 5-methylcytosine during single-molecule, real-time sequencing. Using Tet1 to convert 5-methylcytosine to 5-carboxylcytosine improves the detection rate of this important epigenetic marker, thereby complementing the set of readily detectable microbial base modifications, and enhancing the ability to interrogate eukaryotic epigenetic markers.

PMID:
23339471
PMCID:
PMC3598637
DOI:
10.1186/1741-7007-11-4
[Indexed for MEDLINE]
Free PMC Article
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