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

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
Joe Parker
Early-career Research Fellow (Phylogenomics), Royal Botanic Gardens, Kew
Skype, Twitter, Github: @lonelyjoeparker
Email: joe.parker@kew.org
SMRT / Nanopore sequencing for Plant Pathology
Topics:
• What is it?
• Unique capabilities
• Current work
• Plant health
applications
What is Nanopore sequencing?
Unique capabilities
• Portable
• Real-time (~minutes)
• DNA and RNA
• Selective (‘read-until’)
sequencing
• Very long reads
• Flexible / scalable
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
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.)

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

  • 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. SMRT / Nanopore sequencing for Plant Pathology Topics: • What is it? • Unique capabilities • Current work • Plant health applications
  • 3. What is Nanopore sequencing?
  • 4. Unique capabilities • Portable • Real-time (~minutes) • DNA and RNA • Selective (‘read-until’) sequencing • Very long reads • Flexible / scalable
  • 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. 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. 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. 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. GCPP Applications • Cryptic parasite / pathogen spp. discovery • Real-time monitoring of plant health • Import / export sequencing
  • 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

  1. 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
  2. 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)
  3. Examples of the