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Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands.

Authors
  • J, Schreiber
  • Zl, Wescoe
  • R, Abu-Shumays
  • Jt, Vivian
  • B, Baatar
  • Kevin Karplus
  • M, Akeson
Type
Published Article
Journal
Proceedings of the National Academy of Sciences
Publisher
Proceedings of the National Academy of Sciences
Volume
110
Issue
47
Identifiers
DOI: 10.1073/pnas.1310615110
Source
UCSC Bioinformatics biomedical-ucsc
License
Unknown

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.

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