Decoding long nanopore sequencing reads of natural DNA

Laszlo, Andrew H.; Derrington, Ian M.; Ross, Benjamin; Brinkerhoff, Henry; Adey, Andrew; Nova, Ian C.; Craig, Jonathan M.; Langford, Kyle W.; Samson, Jenny Mae; Daza, Riza M.; Doering, Kenji; Shendure, Jay; Gundlach, Jens H.

doi:10.1038/nbt.2950

Cited by 361 publications

(425 citation statements)

References 31 publications

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“…In accordance with the physical dimensions for four nucleotides, we observe a largest current drop for dGMP, centred at 0.8 nA and a smallest current drop for the smallest single nucleotide dCMP, centred at 0.3 nA. These observations are in good agreement with the results obtained for single nucleotide discrimination using protein pores [1][2][3] . Although the current drop for dAMP is slightly larger than that for dTMP (0.65 nA, compared with 0.45 nA), we believe that this inconsistency might be due to the stronger Bmim + affinity towards dAMP compared with dTMP 40 .…”

Section: Identification Of Single Nucleotidessupporting

confidence: 82%

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Identification of single nucleotides in MoS2 nanopores

et al. 2015

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The size of the sensing region in solid-state nanopores is determined by the size of the pore and the thickness of the pore membrane, so ultrathin membranes such as graphene and single-layer molybdenum disulphide could potentially offer the necessary spatial resolution for nanopore DNA sequencing. However, the fast translocation speeds (3,000-50,000 nt ms -1 ) of DNA molecules moving across such membranes limit their usability. Here, we show that a viscosity gradient system based on room-temperature ionic liquids can be used to control the dynamics of DNA translocation through MoS 2 nanopores. The approach can be used to statistically detect all four types of nucleotide, which are identified according to current signatures recorded during their transient residence in the narrow orifice of the atomically thin MoS 2 nanopore. Our technique, which exploits the high viscosity of room-temperature ionic liquids, provides optimal single nucleotide translocation speeds for DNA sequencing, while maintaining a signal-to-noise ratio higher than 10. Single nucleotide identification 1,2 and DNA sequencing 3 have already been demonstrated with biological nanopores. However, the fragility of such pores, together with difficulties related to measuring their pA-range ionic currents and their dependence on biochemical reagents, means that solid-state nanopores remain an attractive alternative 4 . In contrast to biological pores, solid-state nanopores can operate in various liquid media and pH conditions, their production is scalable and compatible with nanofabrication techniques 5 , and they do not require the excessive use of biochemical reagents. These advantages are expected to make solid-state nanopore sequencing cheaper than sequencing with biological pores.The basic sensing principle in sold-state nanopores is the same as in biological pores. A DNA molecule is threaded through a nanometre-sized pore under an applied potential and, ideally, the sequence of nucleotides is read by monitoring small changes in the ionic current flowing through the pore that are caused by individual nucleotides temporarily residing within the pore 6 . However, solid-state nanopores also allow a transverse detection scheme, which is based on detecting changes in the electrical conductivity of a thin semiconducting channel 4 . In both biological and solid-state nanopores, achieving an optimal translocation speed remains a significant challenge , ref. 8) and are also limited by their use of enzymes. Therefore, to achieve genome sequencing, thousands-or even millons-of biological pores need to be integrated into one sequencer. In solid-state nanopores, translocation speeds are on the order of 3,000-50,000 nt ms , the large range being a result of factors such as the pore size (1.5-25 nm) and the applied potential (100-800 mV) 9 . These fast speeds limit the ability of solid-state nanopores to reach single-nucleotide resolution, and are a major obstacle to the use of solid-state nanopores in the sequencing of DNA.Solid-state nanopores also face proble...

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Section: Identification Of Single Nucleotidessupporting

confidence: 82%

“…ingle nucleotide identification 1,2 and DNA sequencing 3 have already been demonstrated with biological nanopores. However, the fragility of such pores, together with difficulties related to measuring their pA-range ionic currents and their dependence on biochemical reagents, means that solid-state nanopores remain an attractive alternative 4 .…”

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confidence: 99%

Identification of single nucleotides in MoS2 nanopores

et al. 2015

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“…Combining the MspA with the phi29 DNA polymerase can slow the translocation of DNA through the nanopore and subsequently sequence 50-nt-long DNA fragments ( figure 3(a)) [19]. Recent advances in signal processing have extended this approach to 5-kilobase-long DNA strands [20]. Another critical parameter in this technique is the translocation velocity [21], which has to be optimized to allow sufficient time for the acquisition of enough data points to determine the level and hence the nucleotide.…”

Section: Next-generation Sequencing Methodsmentioning

confidence: 99%

The emergence of nanopores in next-generation sequencing

Steinbock¹,

Radenović

2015

Nanotechnology

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Next-generation sequencing methods based on nanopore technology have recently gained considerable attention, mainly because they promise affordable and fast genome sequencing by providing long read lengths (5 kbp) and do not require additional DNA amplification or enzymatic incorporation of modified nucleotides. This permits health care providers and research facilities to decode a genome within hours for less than $1000. This review summarizes past, present, and future DNA sequencing techniques, which are realized by nanopore approaches such as those pursued by Oxford Nanopore Technologies.

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“…Nanopore-based sequencing (14) is such a method, although it has been technically challenging to develop and only very recently has permitted some sequence determination of an actual genome (15,16) The basic idea dates back some 20 y and draws from fundamental work in surface science, molecular biology, nanofabrication, and electrochemistry (17). In one manifestation, two aqueous electrolyte solutions are connected through a single protein nanopore, such as such as Mycobacterium smegmatis porin A (MspA).…”

Section: Genomics and Nanotechnologymentioning

confidence: 99%

Nanotechnologies for biomedical science and translational medicine

Heath

2015

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

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In 2000 the United States launched the National Nanotechnology Initiative and, along with it, a well-defined set of goals for nanomedicine. This Perspective looks back at the progress made toward those goals, within the context of the changing landscape in biomedicine that has occurred over the past 15 years, and considers advances that are likely to occur during the next decade. In particular, nanotechnologies for health-related genomics and single-cell biology, inorganic and organic nanoparticles for biomedicine, and wearable nanotechnologies for wellness monitoring are briefly covered.nanotechnology | biotechnology | nanomedicine

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Decoding long nanopore sequencing reads of natural DNA

Cited by 361 publications

References 31 publications

Identification of single nucleotides in MoS2 nanopores

Identification of single nucleotides in MoS2 nanopores

The emergence of nanopores in next-generation sequencing

Nanotechnologies for biomedical science and translational medicine

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