title: Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands.
abstract: Cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine were identified during translocation of single DNA template strands through a modified Mycobacterium smegmatis porin A (M2MspA) nanopore under control of phi29 DNA polymerase. This identification was based on three consecutive ionic current states that correspond to passage of modified or unmodified CG dinucleotides and their immediate neighbors through the nanopore limiting aperture. To establish quality scores for these calls, we examined ~3,300 translocation events for 48 distinct DNA constructs. Each experiment analyzed a mixture of cytosine-, 5-methylcytosine-, and 5-hydroxymethylcytosine-bearing DNA strands that contained a marker that independently established the correct cytosine methylation status at the target CG of each molecule tested. To calculate error rates for these calls, we established decision boundaries using a variety of machine-learning methods. These error rates depended upon the identity of the bases immediately 5' and 3' of the targeted CG dinucleotide, and ranged from 1.7% to 12.2% for a single-pass read. We estimate that Q40 values (0.01% error rates) for methylation status calls could be achieved by reading single molecules 5-19 times depending upon sequence context.
authors.affiliation: Nanopore Group, Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064.
authors.forename: Jacob
authors.initials: J
authors.lastname: Schreiber
authors.forename: Zachary L
authors.initials: ZL
authors.lastname: Wescoe
authors.forename: Robin
authors.initials: R
authors.lastname: Abu-Shumays
authors.forename: John T
authors.initials: JT
authors.lastname: Vivian
authors.forename: Baldandorj
authors.initials: B
authors.lastname: Baatar
authors.forename: Kevin
authors.initials: K
authors.lastname: Karplus
authors.forename: Mark
authors.initials: M
authors.lastname: Akeson
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry / Q000302:isolation & purification* / Q000031:analogs & derivatives; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification*; D004247:DNA / Q000032:analysis*; D019175:DNA Methylation / Q000235:genetics*; D057890:Epigenomics / Q000379:methods*; D058608:Nanopores; D012107:Research Design
chemical_list: C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA
keywords: MspA; epigenetics