Nanopore-Based Identification of Individual Nucleotides for Direct RNA Sequencing

Ayub, Muhammad Adnan; Hardwick, Steven W.; Luisi, Ben F.; Bayley, Hagan

doi:10.1021/nl403469r

Cited by 109 publications

(114 citation statements)

References 53 publications

(122 reference statements)

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“…One is where each nucleotide is read while the RNA molecule is threaded through the pore. The second strategy is RNA exosequencing where RNA is successively cleaved using polynucleotide phosphorylase, whereafter each nucleotide is read separately in the nanopore (Ayub et al, 2013). Dual RNA-seq of infected single cells together with an intracellular bacterial pathogen was demonstrated with the latter approach (Hardwick et al, 2011).…”

Section: Next Generation Of Dual Approachesmentioning

confidence: 99%

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

Naidoo¹,

Visser²,

Zwart³

et al. 2018

Current Issues in Molecular Biology

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RNA-sequencing technology has been widely adopted to investigate host responses during infection with pathogens. Dual RNA-sequencing (RNA-seq) allows the simultaneous capture of pathogen-specific transcripts during infection, providing a more complete view of the interaction. In this review, we focus on the design of dual RNAseq experiments and the application of downstream data analysis to gain biological insight into both sides of the interaction. Recent literature in this area demonstrates the power of the dual RNA-seq approach and shows that it is not limited to model systems where genomic resources are available. Sequencing costs continue to decrease and single cell transcriptomics is becoming more feasible. In combination with proteomics and metabolomics studies, these technological advances are likely to contribute to our understanding of the temporal and spatial aspects of dynamic plant-pathogen interactions. A dual approach in plantaThe interaction between plants and pathogens is an active and dynamic process that can be likened to a duel. Plants have complex defence mechanisms that can be rendered ineffective when pathogens interfere with one of the various processes required for host defence. These processes include penetration resistance, recognition by Pattern Recognition Receptors (PRRs), phytohormone signalling pathways, secretory pathways, secondary metabolite production, and plant cell death (Dou and Zhou, 2012). Until recently, transcriptomic approaches have been applied in the host and pathogen separately to obtain the gene expression profile of each organism and gain insight into infection biology or host defence mechanisms.RNA sequencing (RNA-seq) is a powerful technology that does not rely on any prior knowledge of transcripts and can generate vast quantities of data with much smaller costs involved than for older techniques such as microarrays (Pareek et al., 2011;Wilhelm and Landry, 2009). An advantage of RNA-seq in the field of plant-pathogen interactions is that both plant and pathogen transcripts can be detected simultaneously and accurately in the same sample. This tactic, known as dual RNAseq, in planta RNA-seq, simultaneous RNA-seq, or comparative RNA-seq, is a relatively new technique both in the plant and medical fields. In plants, it allows for the study of plant-pathogen interactions in herbaceous crops Kunjeti et al., 2012;Lowe et al., 2014) as well as trees (Hayden et al., 2014;Liang et al., 2014;Teixeira et al., 2014). This review outlines technical considerations for dual RNA-seq experiments, summarizes recent insights drawn from such approaches in plant-pathogen interactions, and provides an

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Section: Next Generation Of Dual Approachesmentioning

confidence: 99%

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

Naidoo¹,

Visser²,

Zwart³

et al. 2018

Current Issues in Molecular Biology

View full text Add to dashboard Cite

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“…The first avian transcriptomes have recently been sequenced using this new technology (Kuo et al 2017;Workman et al 2017). Another new application, which enables sequencing of full-length transcripts, is the MinION from Oxford Nanopore Technologies (Ayub et al 2013;Garalde et al 2016). However, to our knowledge this application has not yet been used for RNA sequencing in bird species.…”

Section: Choice Of Sequencing Platformmentioning

confidence: 99%

“…Also, most current RNA-seq protocols contain a templateamplification step, and as certain transcripts might be more prone to amplification than others, this step can lead to the uneven distribution of transcripts which is not representative of the sample (Garalde et al 2016;Kozarewa et al 2009). New technologies are continuously being developed to avoid or minimise these issues, including long-read technologies (Gonzalez-Garay 2016), direct RNA sequencing technologies (Ayub et al 2013;Garalde et al 2016;Ozsolak et al 2009), and PCR-free protocols (Mamanova et al 2010). Ensuring a high -quality library is of highest priority in all NGS projects, and it is therefore likely that we will see further developments aimed at minimising the problems involved in library preparation for RNA-seq within the near future.…”

Section: Library Preparation: Possible Bias In the Datamentioning

confidence: 99%

Avian transcriptomics: opportunities and challenges

Jax

Kraus

2018

J Ornithol

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Recent developments in next-generation sequencing technologies have greatly facilitated the study of whole transcriptomes in model and non-model species. Studying the transcriptome and how it changes across a variety of biological conditions has had major implications for our understanding of how the genome is regulated in different contexts, and how to interpret adaptations and the phenotype of an organism. The aim of this review is to highlight the potential of these new technologies for the study of avian transcriptomics, and to summarise how transcriptomics has been applied in ornithology. A total of 81 peer-reviewed scientific articles that used transcriptomics to answer questions within a broad range of study areas in birds are used as examples throughout the review. We further provide a quick guide to highlight the most important points which need to be take into account when planning a transcriptomic study in birds, and discuss how researchers with little background in molecular biology can avoid potential pitfalls. Suggestions for further reading are supplied throughout. We also discuss possible future developments in the technology platforms used for ribonucleic acid sequencing. By summarising how these novel technologies can be used to answer questions that have long been asked by ornithologists, we hope to bridge the gap between traditional ornithology and genomics, and to stimulate more interdisciplinary research.

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“…It involves DNA strand synthesis from single-stranded (ss) to double-stranded (ds) facilitated by a DNA polymerase such as enzyme phi29 DNA polymerase [33,89,90] or polynucleotide phosphorylase. [91] The translocation becomes stepwise and the DNA strand move one base at a time in accordance to the completion of the strand synthesis from ss to ds. This strategy forms the base for several commercial sequencing technologies such as semiconductor genome sequencing [33] and zero-mode waveguides.…”

Section: Way Forwardmentioning

confidence: 99%

On nanopore DNA sequencing by signal and noise analysis of ionic current

et al. 2016

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Scheicher, R. et al. (2016) On nanopore DNA sequencing by signal and noise analysis of ionic current. Our focus is placed on signal-boosting and noise-suppressing strategies in order to attain the single-nucleotide resolution. Apart from decreasing pore diameter and thickness, it is crucial to also reduce the translocation speed and facilitate a stepwise translocation. Our best-case scenario analysis points to severe challenges with employing plain nanopore technology, i.e., without recourse to any signal amplification strategy, in achieving sequencing with the desired single-nucleotide resolution. A conceptual approach based on strand synthesis in the nanopore of the translocating DNA from single-stranded to double-stranded is shown to yield a 10-fold signal amplification. Although it involves no advanced physics and is very simple in mathematics, this simple model captures the essence of nanopore sequencing and is useful in guiding the design and operation of nanopore sequencing. Nanotechnology

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Nanopore-Based Identification of Individual Nucleotides for Direct RNA Sequencing

Cited by 109 publications

References 53 publications

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

Avian transcriptomics: opportunities and challenges

On nanopore DNA sequencing by signal and noise analysis of ionic current

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