This document summarizes a study that used nanopore sequencing to analyze plant and microbial DNA from oak trees affected by Acute Oak Decline (AOD) at multiple sites in the UK. Key findings include: (1) Potential bacterial "vectors" of AOD were detected at nearly all sites in both symptomatic and asymptomatic trees; (2) Differences in bacterial communities between bark and leaf samples were unclear; (3) Non-native oak species were also affected; (4) Challenges remained in data analysis and establishing microbial baselines. The study demonstrated the feasibility of field-based metagenomic sequencing to identify pathogens.

1. Field-based, real-time metagenomics and
phylogenomics for responsive pathogen
detection: lessons from nanopore analyses
of Acute Oak Decline (AOD) sites in the UK
Joe Parker
Early-career Research Fellow, RBG Kew
@lonelyjoeparker:
joe.parker@kew.org

2. Acute Oak Decline
Acute oak decline:
A syndrome-type oak disease
• Unknown cause, no treatment
• ca. 200 million oaks in GB
…amenity & timber value: ~£500/tree
• Emerged ca. 2004, spreading rapidly
• Significant morbidity and mortality
Defra ‘Futureproofing Plant Health’ initiative
• Test field-based methods
• Balanced survey of microbial community
composition (healthy & affected individuals)
• Overcome ascertainment bias
• Pilot training of non-experts.
• Draw conclusions relevant to rapid-response plant
health monitoring in the UK.
© 2016 Katy Reed / Forest Research

3. Sites / samples
• Three sites: Kew, Richmond, Malvern Wood
• Symptomatic and asymptomatic
• Several species – not just native (Kew)
• Incl. N America, Near East, China/SE Asia
• Tissues: bark, leaf, exudate
• In silica and also straight to -20ºC
L-R: Q. petraea; Q. frainetto; Q. nigra

4. Adventures with DNA
sequencing

6. Field-sequencing for real
Conditions
100% humidity; 6-13ºC
Essential kit
800w generator
3x laptops
Centrifuge
Waterbath
Polystyrene boxes (lots)
Kettle(…!)
Yield
>400Mbp data in three days;
A. thaliana ~2.01x coverage

7. Key targets found?
Target Symptomatic Asymptomatic
A. biggutatus ~5% ~4%
Brennaria sp. 1% ~1%
Gibbsiella sp. <<1% ~0%
Rahnella sp. 0% ~0%

8. Kraken (WIMP)
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
SYM - various
ASYM*
SYM - Various incl. exudate
SYM - Kew, non-native
SYM - Richmond, bark Aspergillaceae
Chaetomiaceae
Clavicipitaceae
Cordycipitaceae
Debaryomycetaceae
Glomerellaceae
Herpotrichiellaceae
Hypocreaceae
Leptosphaeriaceae
Phycomycetaceae
Pleosporaceae
Pucciniaceae
Saccharomycetaceae
Sordariaceae

9. Challenges – informatics

10. Partial queries: encounter curves?
Subsample MinION output
Repeat ID pipeline, record
mean ID stat sbias
Replicates: N = 30
Simulate from 100 – 104 reads
(≈instant → hours)

11. Partial references?
Take reference genome at
high contiguity
Fragment randomly to
target (low) contiguity
Repeat read identification
using fragmented DB
Simulate N50 ≈1,000bp
to N50 ≈ 10Mbp

12. Long-read matching vs K-mers
Entire chloroplast
genome (~150kbp)
Plastid
coding loci
Individual field-
sequenced
MinION reads

13. Gene prediction – phylogenomics and functional descriptions?
Filtered
reads
Gene
models
TAIR10
CDS code
Annotation
SNAP
1:1 reciprocal
BLAST
Multiple sequence
alignments
MUSCLE
Trimal
Gene trees → Consensus tree
*BEAST
RAxML,
TreeAnnotator
Cumulative counts:
Unique genes
All genes
(‘Lab’ being
transported!)

14. (Early) AOD conclusions
• ‘Vector’ sequences almost ubiquitous
• Bark / leaf differences unclear
• Non-native species affected
• ‘Polysyndromic’ bacteria?
• Relative abundance?
• Microbiome not understood – baseline
data
• Temperature/climate effect?

15. Field-based ID systems
• Data is decent
• Need good long-read-aware + noise
correction
• More data to establish baselines
• Mainly expertise/know-how limited, tech
is fine now.

16. Ubiquitous / citizen-sequencing
From lab-based…
… to ‘app store’ genomics

17. Keeping it simple: Kew Science Festival
Six species: whole genome-
skim samples with MinION
in preparation
Build BLAST DBs from
skimmed data
Select ‘unknown’ (blinded)
sample, extract DNA and
resequence in real-time
Compare to partial DBs in
six-way BLAST competition
Live ID ?

18. Thanks, funders, contacts and questions
Oxford Nanopore
Technologies Ltd.
Dan Turner, Richard
Ronan, Gerrard CoyneU Bangor:
Alexander S.T. Papadopulos (@metallophyte)
RBG Kew:
Postdocs: Andrew Helmstetter (@ajhelmstetter); Tim Coker
Thanks: Dion Devey, Robyn Cowan, Tim Wilkinson, Stephen Dodsworth, Pepijn Kooij, Felix
Forest, Bill Baker, Jan T. Kim, Jenny Williams, Abigail Barker, Mark Lee, Jim Clarkson, Mike
Chester, Ester Gaya, Lisa Pokorny, Laszlo Csiba, Paul Wilkin, Richard Buggs, Mike Fay, Mark
Chase, Ilia Leitch
QMUL
Laura Kelly, Kalina Davies, Steve Rossiter
Oxford
Aris Katzourakis, Oli Pybus, Jayna Raghwani
Others
Forest Research: Daegan Inward, Katy Reed
Dstl: Claire Lonstale, James Taylor
Birmingham: Nick Loman, Josh Quick
U. Utah: Bryn Dentinger
Imperial: James Rosindell
This research was
conducted in the
Sackler Phylogenomics
Laboratory and was
supported by the
Calleva Foundation
Phylogenomic Research
Programme and the
Sackler Trust
@lonelyjoeparker:
joe.parker@kew.org