Replication of individual DNA molecules under electronic control using a protein nanopore

Olasagasti, Felix; Lieberman, Kate R.; Benner, Seico; Cherf, Gerald Maxwell; Dahl, Joseph M.; Deamer, David W.; Akeson, Mark

doi:10.1038/nnano.2010.177

Cited by 161 publications

(154 citation statements)

References 24 publications

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“…shown in Fig. 2A, along with an annealed blocking oligomer that protected the 3′-OH terminus from phi29 DNAP-catalyzed strand elongation in bulk phase (16,20). The single-stranded 59-nt overhang of the DNA substrate contained the CG dinucleotide that is the focus of this study.…”

Section: Resultsmentioning

confidence: 99%

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

Schreiber

Wescoe

Abu-Shumays

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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172

156

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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...

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Section: Resultsmentioning

confidence: 99%

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

Schreiber

Wescoe

Abu-Shumays

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

172

156

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“…The technology closest to the market is exonuclease-based[121] and will be marketed, sold, distributed, and serviced by Illumina. The alternative technology, termed ‘strand sequencing’ [124] may be capable of rereading the same strand multiple times. It remains to be seen what the throughput and error rate of the instruments will be, but due to a lower reagent cost and the absence of a library preparation, the cost of the assay will most likely be significantly lower than current technologies.…”

Section: Future Prospectsmentioning

confidence: 99%

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Gundry

Vijg

2012

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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DNA mutations are the source of genetic variation within populations. The majority of mutations with observable effects are deleterious. In humans mutations in the germ line can cause genetic disease. In somatic cells multiple rounds of mutations and selection lead to cancer. The study of genetic variation has progressed rapidly since the completion of the draft sequence of the human genome. Recent advances in sequencing technology, most importantly the introduction of massively parallel sequencing (MPS), have resulted in more than a hundred-fold reduction in the time and cost required for sequencing nucleic acids. These improvements have greatly expanded the use of sequencing as a practical tool for mutation analysis. While in the past the high cost of sequencing limited mutation analysis to selectable markers or small forward mutation targets assumed to be representative for the genome overall, current platforms allow whole genome sequencing for less than $5,000. This has already given rise to direct estimates of germline mutation rates in multiple organisms including humans by comparing whole genome sequences between parents and offspring. Here we present a brief history of the field of mutation research, with a focus on classical tools for the measurement of mutation rates. We then review MPS, how it is currently applied and the new insight into human and animal mutation frequencies and spectra that has been obtained from whole genome sequencing. While great progress has been made, we note that the single most important limitation of current MPS approaches for mutation analysis is the inability to address low-abundance mutations that turn somatic tissues into mosaics of cells. Such mutations are at the basis of intra-tumor heterogeneity, with important implications for clinical diagnosis, and could also contribute to somatic diseases other than cancer, including aging. Some possible approaches to gain access to low-abundance mutations are discussed, with a brief overview of new sequencing platforms that are currently waiting in the wings to advance this exploding field even further.

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“…Combination of a-haemolysin with processive enzymes was recently demonstrated [54,55], so this approach holds great promise for DNA sequencing. It is even possible to map the extension of DNA templates with 0.5 nm resolution [56][57][58]. This is another indication of the versatility of the nanopores sensing approach and its great sensitivity for changes on molecular length scales.…”

Section: Modification Of Biological Nanoporesmentioning

confidence: 99%

Controlling molecular transport through nanopores

Keyser

2011

J. R. Soc. Interface.

173

191

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Nanopores are emerging as powerful tools for the detection and identification of macromolecules in aqueous solution. In this review, we discuss the recent development of active and passive controls over molecular transport through nanopores with emphasis on biosensing applications. We give an overview of the solutions developed to enhance the sensitivity and specificity of the resistive-pulse technique based on biological and solid-state nanopores.

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Replication of individual DNA molecules under electronic control using a protein nanopore

Cited by 161 publications

References 24 publications

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

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

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Controlling molecular transport through nanopores

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