Continuous base identification for single-molecule nanopore DNA sequencing

Clarke, James; Wu, Hai‐Chen; Jayasinghe, Lakmal; Patel, Aavishkar A.; Reid, S.; Bayley, Hagan

doi:10.1038/nnano.2009.12

Cited by 1,529 publications

(1,349 citation statements)

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“…3)[63, 120]. Oxford Nanopore’s sequencing platform uses an exonuclease cleavage reaction and a protein nanopore to sequence individual cleaved bases by a unique electrical signature as they are transported through the pore[121]. …”

Section: Future Prospectsmentioning

confidence: 99%

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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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Section: Future Prospectsmentioning

confidence: 99%

“…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.…”

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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“…More broadly, the growing library of methods to manipulate DNA molecules in nanofluidic devices has enabled fundamental research about single polymer molecules. 30,31 These studies inform important applications such as genomic sequencing via nanopore translocation 32 or direct linear analysis. 33 Thus, (un)tying knots in polymers is of interest in its own right.…”

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confidence: 99%

Untying Knotted DNA with Elongational Flows

Renner

Doyle

2014

ACS Macro Lett.

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We present Brownian dynamics simulations of initially knotted double-stranded DNA molecules untying in elongational flows. We show that the motions of the knots are governed by a diffusion−convection equation by deriving scalings that collapse the simulation data. When being convected, all knots displace nonaffinely, and their rates of translation along the chain are topologically dictated. We discover that torus knots "corkscrew" when driven by flow, whereas nontorus knots do not. We show that a simple mechanism can explain a coupling between this rotation and the translation of a knot, explaining observed differences in knot translation rates. These types of knots are encountered in nanoscale manipulation of DNA, occur in biology at multiple length scales (DNA to umbilical cords), and are ubiquitous in daily life (e.g., hair). These results may have a broad impact on manipulations of such knots via flows, with applications to genomic sequencing and polymer processing.K nots are commonly encountered and manipulated in everyday experiences such as tying one's shoelaces or untangling spontaneously knotted strings. 1 Formally defined only for closed rings, the topologies of "open" knots (referred to hereafter simply as knots) are often unambiguous (e.g., shoelaces and neckties) and can be closed and algorithmically defined. 2−4 At microscopic scales, chromosomal knots are modified by topoisomerases during cell division 5 and are thought to participate in gene regulation. 6 Knots are found in proteins 7,8 and viral capsid DNA,9,10 likely with yet to be fully understood functions. It has been mathematically proven that knots become asymptotically likely as the length of a polymer increases, 11 a fact that explains their ubiquity.Due to the emerging significance of knotted polymers, a growing body of simulation literature is devoted to their study. 12,13 For instance, while the topology of a ring is fixed, an open polymer can spontaneously form and untie knots. 14 The probability of forming such knots can be nonmonotonic when the polymer is confined in slits 15,16 or tubes, 17 and increasing the stiffness of a polymer can similarly influence the knotting probability in unintuitive ways. 18,19 Such knots can substantially affect the mechanical properties 20,21 and rheological behavior 22 of a polymer, and the probability of forming knots has been used to infer the effective diameter of DNA molecules. 23 Recently, simulations have shown dramatic slowing of processes wherein a knot is driven along a chain such as entropic ejection of DNA from a viral capsid 24 and the translocation of single-stranded DNA (ssDNA) 25 and polypeptides 26 through pores.Common nanofluidic experiments have led to the spontaneous formation of knots in DNA by collision with channel defects 27 or the application of moderate electric fields 28,29 during electrophoresis. More broadly, the growing library of methods to manipulate DNA molecules in nanofluidic devices has enabled fundamental research about single polymer molecules. 30,31 Th...

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“…These approaches also have the potential to identify unknown cofactors of a protein complex. When combined with single-molecule ELISA (enzyme-linked immunosorbent assay) [57], single-molecule sequencing technologies [58][59][60], single-molecule multi-color FRET (fluorescence resonance energy transfer) [61][62][63], and screening tools, the new techniques discussed in this review will acquire versatility and be used for practical analysis in the near future, complementing mass spectroscopy techniques.…”

Section: Discussionmentioning

confidence: 99%

Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. Single-molecule protein studiesSingle-molecule techniques have become potent tools for the discovery of novel protein mechanisms by allowing high spatiotemporal resolution. Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.Among these techniques, single-molecule fluorescence spectroscopy has enabled researchers to unveil the action mechanism of a protein by imaging protein activity in real time. Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] Single-molecule observations using cell extractsUnlike purified recombinant proteins, crude cell extracts provide more biologically relevant environment in which molecules of interest can interact not only with their partners but also with other cellular components. It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al. used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al. immunostained reaction products using specific fluorescent antibodies [15,20]. Real-time observation of spliceosome assemblyDespite the relatively small number of genes in the human genome, we have extraordin...

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Continuous base identification for single-molecule nanopore DNA sequencing

Cited by 1,529 publications

References 32 publications

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

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

Untying Knotted DNA with Elongational Flows

Bringing single-molecule spectroscopy to macromolecular protein complexes

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