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.MspA | epigenetics E pigenetic modifications of DNA help regulate gene transcription in biological cells. In mammals, 5-methylcytosine (mC) modification of CG dinucleotides is known to influence development (1, 2) and contribute to human diseases including cancer (3). Other modifications have been detected at carbon 5 of cytosine including 5-hydroxymethylcytosine (hmC) (4), and more recently 5-formylcytosine, and 5-carboxycytosine (5). Physiological roles for hmC in carcinogenesis and embryonic stem cell differentiation have been proposed (6).High-throughput techniques for mC detection are based on bisulfite treatment of genomic DNA (7). In the conventional assay, cytosine (but not mC nor hmC) is converted to uracil (8). Thus, positions not converted to uracil identify cytosines that were modified in the original genomic sequence. In a landmark paper, Lister et al. (9) used this technique to map genome-wide cytosine methylation in human embryonic stem cells and fetal lung fibroblasts at single-nucleotide precision. Recently, bisulfite strategies for discriminating between mC and hmC using the Tet1 enzyme (10) or by chemical modification of hmC (11) have been described.Single-molecule techniques have emerged as possible alternatives to bisulfite treatment for detecting epigenetic modifications of DNA (12). These single-molecule approaches share several useful features including few processing steps before sequence analysis, long reads that routinely exceed several thousand nucleotides, and the ability to read native DNA strands in heterogeneous mixtures. The most advanced of these single-molecule techniques, from Pacific Biosciences, uses fluorescence to detect labeled nucleotide triphosphates during daughter-strand elongation. This elongation is catalyzed by a DNA pol...

Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome

“…This is likely due to the challenges of the signal processing the ionic current measurements (Schreiber et al 2013) as well as the challenges inherent in any type of single-molecule sequencing. Oxford Nanopore has indicated that the pores are more than a single base in height so that the ionic signal measurements are not of individual nucleotides but of ∼5 nt at a time.…”

Alignathon: a competitive assessment of whole-genome alignment methods

“…The starting point of the Alignathon is to assume that the problem of genome assembly is largely a solved problem. Although we admit this is currently a dubious assumption, it appears that the problem of genome assembly will shrink in size in the coming years as new sequencing technologies become available and existing assembly software is perfected to take advantage of more numerous, longer, and less error-prone reads (Branton et al 2008;Schreiber et al 2013;Laszlo et al 2014). With this future as a starting point, the question a biologist faces changes from a proximate one of ''how do I best assemble the genome of my favorite species?''…”

Review of Clinical Next-Generation Sequencing

“…114 As the structure (DNA or RNA strand) passes through the pore, the current changes and the degree of change correlates with the individual base (A, C, G, or T) and also correlates with the methylation status of C; therefore, methylation and hydroxymethylation can be detected. 114,116 The PacBio SMRT can also infer methylation status by analyzing the change in DNA polymerase kinetics (the time to incorporate a base and the time between incorporation of 2 bases).…”