Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation
1 Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA
2 Department of Chemistry and Institute for Biophysical Dynamics, 929 East 57th Street, The University of Chicago, Chicago, Illinois, 60637, USA
BMC Biology 2013, 11:4 doi:10.1186/1741-7007-11-4Published: 22 January 2013
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.
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.
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.