The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community

Jain, Miten; Olsen, Hugh E.; Paten, Benedict; Akeson, Mark

doi:10.1186/s13059-016-1103-0

Cited by 1,245 publications

(1,044 citation statements)

References 63 publications

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“…Synthetic long read sequencing or thirdgeneration sequencing technologies may allow future studies to detect base pairings involving sequence separations longer than 250 nt (32)(33)(34). In terms of seeding mutations, applications to viruses and other systems that involve high-error rate RNA polymerases may obviate this step, but generally M2-seq in extracts, cells, and tissues will require transfecting DNA or RNA libraries that are prepared through error-prone PCR or other emerging techniques (33,34). A faster and less biologically perturbing protocol would be enabled by a cell-permeable mutagen that could directly attack nucleotides initially sequestered inside RNA helices.…”

Section: Discussionmentioning

confidence: 99%

RNA structure inference through chemical mapping after accidental or intentional mutations

Cheng

Kladwang

Yesselman

et al. 2017

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

104

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Despite the critical roles RNA structures play in regulating gene expression, sequencing-based methods for experimentally determining RNA base pairs have remained inaccurate. Here, we describe a multidimensional chemical-mapping method called "mutate-and-map read out through next-generation sequencing" (M2-seq) that takes advantage of sparsely mutated nucleotides to induce structural perturbations at partner nucleotides and then detects these events through dimethyl sulfate (DMS) probing and mutational profiling. In special cases, fortuitous errors introduced during DNA template preparation and RNA transcription are sufficient to give M2-seq helix signatures; these signals were previously overlooked or mistaken for correlated double-DMS events. When mutations are enhanced through error-prone PCR, in vitro M2-seq experimentally resolves 33 of 68 helices in diverse structured RNAs including ribozyme domains, riboswitch aptamers, and viral RNA domains with a single false positive. These inferences do not require energy minimization algorithms and can be made by either direct visual inspection or by a neural-network-inspired algorithm called M2-net. Measurements on the P4-P6 domain of the Tetrahymena group I ribozyme embedded in Xenopus egg extract demonstrate the ability of M2-seq to detect RNA helices in a complex biological environment.RNA structure modeling | chemical mapping | neural network | mutational profiling | Xenopus egg extract I nference of RNA structures using experimental data is a crucial step in understanding RNA's biological functions throughout living organisms. Chemical-mapping methods have the potential to reveal RNA structural features in situ by probing which nucleotides are protected from attack by chemical modifiers. The resulting experimental data can be used to guide secondary-structure modeling by computational algorithms, raising the prospect of transcriptome-wide RNA structure determination (1, 2).Despite these advances, the accuracy of RNA structure inference through chemical mapping and sequencing remains under question (3-8). For example, models of the 9-kb HIV-1 RNA genome have been repeatedly revised with updates to the selective 2′-OH acylation by primer extension (SHAPE) protocol, data processing, and computational assumptions (2, 9-11), and the majority of this RNA's helices remain uncertain. Even for small RNA domains, SHAPE and dimethyl sulfate (DMS) (methylation of N1 and N3 atoms at A and C) have produced misleading secondary structures for ribosomal domains and blind modeling challenges that have been falsified through crystallography or mutagenesis (3,7,12,13). In alternative approaches based on photoactivated cross-linkers, many helix detections appear to be false positives, based on ribosome data in vitro and in vivo (14,15).The confidence and structural accuracy of chemical-mapping methods can be improved by applying perturbations to the RNA sequence before chemical modification. In the mutate-and-map strategy, mapping not just the target RNA sequence but also a compre...

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Section: Discussionmentioning

confidence: 99%

RNA structure inference through chemical mapping after accidental or intentional mutations

Cheng

Kladwang

Yesselman

et al. 2017

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

104

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“…The MinION directly connects to a laptop or desktop PC via a USB3 port, weighs ∼90 g, and costs are for consumable reagents only. The MinION works on the principle of nanopore strand sequencing in real time Jain et al 2016) and results in sequence read lengths from several hundred bases to hundreds of thousands of bases. To date, most studies have used the MinION to sequence small genomes or to partially survey larger genomes to assess chromosomal structure and copy-number variations (Goodwin et al 2015;Loman et al 2015;Norris et al 2016;Wei and Williams 2016).…”

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confidence: 99%

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

et al. 2017

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Advances in long-read single molecule sequencing have opened new possibilities for ‘benchtop’ whole-genome sequencing. The Oxford Nanopore Technologies MinION is a portable device that uses nanopore technology that can directly sequence DNA molecules. MinION single molecule long sequence reads are well suited for de novo assembly of complex genomes as they facilitate the construction of highly contiguous physical genome maps obviating the need for labor-intensive physical genome mapping. Long sequence reads can also be used to delineate complex chromosomal rearrangements, such as those that occur in tumor cells, that can confound analysis using short reads. Here, we assessed MinION long-read-derived sequences for feasibility concerning: (1) the de novo assembly of a large complex genome, and (2) the elucidation of complex rearrangements. The genomes of two Caenorhabditis elegans strains, a wild-type strain and a strain containing two complex rearrangements, were sequenced with MinION. Up to 42-fold coverage was obtained from a single flow cell, and the best pooled data assembly produced a highly contiguous wild-type C. elegans genome containing 48 contigs (N50 contig length = 3.99 Mb) covering >99% of the 100,286,401-base reference genome. Further, the MinION-derived genome assembly expanded the C. elegans reference genome by >2 Mb due to a more accurate determination of repetitive sequence elements and assembled the complete genomes of two co-extracted bacteria. MinION long-read sequence data also facilitated the elucidation of complex rearrangements in a mutagenized strain. The sequence accuracy of the MinION long-read contigs (∼98%) was improved using Illumina-derived sequence data to polish the final genome assembly to 99.8% nucleotide accuracy when compared to the reference assembly.

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“…Les modèles d'inférences statistiques (prédiction probabiliste) actuels décomposent le signal en unités glissantes (ou fenêtres mobiles) de 5 bases (5-mers) reproduisant la longueur du fragment piégé dans la constriction (par des approches statistiques [14] et bioinformatiques [15,16]). À l'issue de ce processus d'amélioration, cette technique, commercialisée par Oxford Nanopore Technologies [17,48] (➜) et fondée sur des pores biologiques et la détec-tion électrique, permet d'extraire la séquence de longs fragments individuels de 10 à 1 000 kb, ce qui autorise la compensation par redondance d'un taux d'erreur total élevé. Le taux d'erreur brut total est, dans le meilleur des cas, de 7,5 % pour la lecture d'une base individuelle chez E. coli, en comptant erreurs, insertions et délétions [18].…”

Section: Séquençage De L'adn Par Nanopores Résultats Et Perspectivesunclassified

“…Des protocoles ont été développés pour augmenter la reproductibilité de ces mesures [32]. Des comparaisons avec les autres techniques de séquençage couramment utilisées ont pu montrer que cette méthode est aujourd'hui compétitive pour le séquençage de novo et l'analyse de variants [17,18,33,34]. Aujourd'hui, les recherches académiques et industrielles cherchent à étendre le principe de séquençage par nanopore à d'autres biomolé-cules, en particulier aux polypeptides [35].…”

Section: Séquençage De L'adn Par Nanopores Résultats Et Perspectivesunclassified

Séquençage de l’ADN par nanopores

Montel

2018

Med Sci (Paris)

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Après des années de développement, l’utilisation du nanopore comme sonde pour séquencer les molécules d’ADN est maintenant une possibilité viable et prometteuse. La détection d’une seule paire de bases lors du transport de l’ADN permet d’enregistrer de très longs fragments de polynucléotides, avec une parallélisation et des vitesses élevées. Dans cette revue, les méthodologies actuelles fondées sur la détection électrique et les nanopores biologiques seront présentées de même que les nouvelles méthodes utilisant des nanopores à l’état solide, ou la détection optique.

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The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community

Cited by 1,245 publications

References 63 publications

RNA structure inference through chemical mapping after accidental or intentional mutations

RNA structure inference through chemical mapping after accidental or intentional mutations

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

Séquençage de l’ADN par nanopores

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