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Single-molecule real-time (SMRT) Nanopore sequencing for Plant Pathology applications

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Single-molecule real-time (SMRT) Nanopore sequencing for Plant Pathology applications

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A short presentation to the British Society for Plant Pathology's 'Grand Challenges in Plant Pathology' workshop on the uses of real-time DNA/RNA sequencing technology for plant health applications.

Doctoral Training Centre, University of Oxford, 14th September 2016.

A short presentation to the British Society for Plant Pathology's 'Grand Challenges in Plant Pathology' workshop on the uses of real-time DNA/RNA sequencing technology for plant health applications.

Doctoral Training Centre, University of Oxford, 14th September 2016.

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Single-molecule real-time (SMRT) Nanopore sequencing for Plant Pathology applications

  1. 1. Single-molecule real-time (SMRT) Nanopore sequencing for Plant Pathology applications Joe Parker Early-career Research Fellow (Phylogenomics), Royal Botanic Gardens, Kew Skype, Twitter, Github: @lonelyjoeparker Email: joe.parker@kew.org
  2. 2. SMRT / Nanopore sequencing for Plant Pathology Topics: • What is it? • Unique capabilities • Current work • Plant health applications
  3. 3. What is Nanopore sequencing?
  4. 4. Unique capabilities • Portable • Real-time (~minutes) • DNA and RNA • Selective (‘read-until’) sequencing • Very long reads • Flexible / scalable
  5. 5. Real projects (i) Real-time, portable sequencing for ebolavirus (EBV) surveillance, 2015 (Guinea) Local sequencing in primary care, limitations on: • facilities, • internet bandwidth, • computation Data uploaded for further analysis worldwide Sampling-to-answer in ~days Phylogeography, demography and evolution of EBV monitored in real- time Quick et al. (2016) http://dx.doi.org/10.1038/natu re16996
  6. 6. Real projects (ii) Field-based genomics for plants: a) Unsupported sequencing and real- time identification of Arabidopsis spp. in Snowdonia  Complete leaf-to-analysis workflow in the field  Field-sequenced data comparable to lab b) Real-time identification of randomised samples  Six diverse plant species randomised and sequenced in tent at Kew Science Festival  Identification < 1hour  Minimal reference needed Papadopulos & Parker (in prep.)
  7. 7. Real projects (iii) Real-time selective sequencing on nanopore technology • Proportion of desired sequences from WGS is low • SMRT is a real-time platform, why not pick and choose individual DNA molecules? • Phage lambda regions selectively sequenced with software-based method • Any region could be selected for resequencing, and alterations to protocol made during a run Loose et al. (2016) http://dx.doi.org/10.1038/nmet h.3930
  8. 8. Real projects (iv) RNA sequencing: -RNA sequenced directly from sample -Stochiometric / quantitative -Tested on E.coli Major implications for transcriptomics, RNA virus detection Garalde et al. (submitted) http://dx.doi.org/10.1101/0688 09
  9. 9. GCPP Applications • Cryptic parasite / pathogen spp. discovery • Real-time monitoring of plant health • Import / export sequencing
  10. 10. Thanks Collaborators / funders: – Kew Foundation Pilot Study Fund – Drs. Dion Devey, Andrew Helmstetter, Alexander S.T. Papadopulos – Oxford Nanopore Technologies – Snowdonia NPA – Natural Resources Wales Contact: Skype, Twitter, Github: @lonelyjoeparker Email: joe.parker@kew.org

Editor's Notes

  • Nanopore sequencing is not new, but it wasn’t usable before
    Now (fairly) routine
    Technology of sequencing not a particular platform. Platforms include MinION, PromethION etc
    Nucleic acids (DNA/RNA) pass through a nanopore under electrical potential
    Deflections in electrical signal
    Squiggles mapped to bases (‘basecalled’) using machine learning
    Real-time
    Fast (sequences in ~mins)
    Scalable capacity
    Accuracy improving
  • What is unique to nanopore-SMRT over other sequencing?
    Real-time
    Field portable – really!
    Selective sequencing
    Scalable (run-until)
    Really long reads >> 50kb

    (if time):
    What it is not:
    Not as routine or high quality as Illumina (so far)
  • Examples of the

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