title: Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation.
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.
authors: Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA.
authors: Tyson A
authors: TA
authors: Clark
authors: Xingyu
authors: X
authors: Lu
authors: Khai
authors: K
authors: Luong
authors: Qing
authors: Q
authors: Dai
authors: Matthew
authors: M
authors: Boitano
authors: Stephen W
authors: SW
authors: Turner
authors: Chuan
authors: C
authors: He
authors: Jonas
authors: J
authors: Korlach
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism*; D015254:DNA Modification Methylases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D016680:Genome, Bacterial; D007700:Kinetics; D006899:Mixed Function Oxygenases; D010084:Oxidation-Reduction; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D044503:5-Methylcytosine; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; D015254:DNA Modification Methylases