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.

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