title: Nanopores discriminate among five C5-cytosine variants in DNA.
abstract: Individual DNA molecules can be read at single nucleotide precision using nanopores coupled to processive enzymes. Discrimination among the four canonical bases has been achieved, as has discrimination among cytosine, 5-methylcytosine (mC), and 5-hydroxymethylcytosine (hmC). Two additional modified cytosine bases, 5-carboxylcytosine (caC) and 5-formylcytosine (fC), are produced during enzymatic conversion of hmC to cytosine in mammalian cells. Thus, an accurate picture of the cytosine epigenetic status in target cells should also include these C5-cytosine variants. In the present study, we used a patch clamp amplifier to acquire ionic current traces caused by phi29 DNA polymerase-controlled translocation of DNA templates through the M2MspA pore. Decision boundaries based on three consecutive ionic current states were implemented to call mC, hmC, caC, fC, or cytosine at CG dinucleotides in ∼4400 individual DNA molecules. We found that the percentage of correct base calls for single pass reads ranged from 91.6% to 98.3%. This accuracy depended upon the identity of nearest neighbor bases surrounding the CG dinucleotide.
authors.affiliation: Department of Biomolecular Engineering, Baskin School of Engineering, MS SOE2, University of California , Santa Cruz, California 95064, United States.
authors.forename: Zachary L
authors.initials: ZL
authors.lastname: Wescoe
authors.forename: Jacob
authors.initials: J
authors.lastname: Schreiber
authors.forename: Mark
authors.initials: M
authors.lastname: Akeson
mesh_terms: D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D058608:Nanopores
chemical_list: D003596:Cytosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase