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

Schreiber J,Wescoe ZL,Abu-Shumays R,Vivian JT,Baatar B,Karplus K,Akeson M

doi

10.1073/pnas.1310615110

subject

Has Abstract

pub_date

2013-11-19 00:00:00

pages

18910-5

issue

47

eissn

0027-8424

issn

1091-6490

pii

1310615110

journal_volume

110

pub_type

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