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eDNA and Plant Pathogen Metabarcoding
Use of Technology in the Natural Environment
SNH Sharing Good Practice day – June 7 2017
David Cooke Leighton Pritchard & Peter Thorpe Sarah Green
Tree Health and Plant Biosecurity initiative
Terminology
eDNA • Total DNA extracted from organisms in an
environmental sample
• skin, water, soil, air, gut contents, pollen sac, etc....
• Contemporary samples, permafrost, sediments
NGS/HTS • Next Gen sequencing/ High throughput sequencing
• Technology - Illumina, Ion Torrent, Nanopore, SMRT
Metagenomics
• Sequencing all DNA in eDNA sample
By Abizar at English Wikipedia, CC BY-SA 3.0
What is metabarcoding?
• Barcoding is a means of discriminating organisms
based on short DNA sequence differences
Species 1 CCACACTGAGCTAAGGCCTTTAA
Species 2 CCACACAGAGGTAAGGCCATTAA
• Metabarcoding - massive increase in
throughput due to advancing sequencing
technology and reduced prices per base pair
Oxford Nanopore Technologies
eDNA metabarcoding applications
• Specific organisms
• Great crested newt surveys
• Biosecurity – search for quarantine organisms
• Biodiversity inventory - conservation monitoring
• Surveys of fish, fungi, YFO etc
• Research projects
• How does a specific treatment affect biodiversity
Metabarcoding – some pros and cons
• High throughput
• Massively parallel – indexing
• Identifies most species (barcode resolution)
• Identifies organisms that cannot be cultured
• Can share DNA samples – synergy between projects
Some challenges
• Need good sampling and replication
• Will not identify hybrid species
• Error rates and contamination risks – false positive and false
negatives
• Blind acceptance is risky – validation needed
• Data storage and computational biology resources are critical
Method Tool
DNA extraction/ PCR
DNAseq
QC, Trim, Chimera detection
Assemble reads
Error correction Bayes Hammer
Nested PCR
Illumina overlapping reads
Fastqc, Trimmomatic, Vsearch
Flash / PEAR
Convert FQ, FA & Trim primers
Cluster
Seqcrumbs, Biopython
Swarm CD-HIT Vsearch Bowtie
Blastclust
Python: sklearn
Compare clustering
Graphics
Summarise species Python
Identify species found by all methods,
Or what was unique to each method
Leighton Pritchard & Peter Thorpe
Reference database (any)
Coded in Python - will be released on GitHub
Computational biology pipeline
Case Study - Phytophthora
• 167 species – destructive plant pathogens • 16 species on UK plant health risk register
Dave Rizzo Youtube.com California Oak Mortality Task Force
Phytophthora zoospore detection
Sampling water
• Irrigation water
• Water flooded through roots of pot-grown plants
• Rivers
Filtration
• Cellulose acetate filters
• DNA extraction
• PCR - Phytophthora genus specific primers
• Sequencing (high or low throughput)
• Invergowrie Burn (IGB) sampled 56 times (every 2 weeks) over 2 years at a single sample point
• Filter DNA extracted and PCR with Phytophthora specific primers
• Run on Illumina MiSeq with v2 chemistry (896K 250bp reads)
• 30 known and 31 unknown Phytophthora species detected
Past success…
Some species abundant all year
Quantitative when considering frequency of occurrence between samples
Winter months
P. xcambivora lower frequency all year
Winter months
Unknown clade 4 species – summer only
Winter months
Herbaceous downy mildew (nettle) summer only
Mycorrhizal Fungi in Montane Heaths
Dr Andy Taylor – James Hutton Institute, Aberdeen
Arctostaphylos alpinus
Salix herbecea
Arctostaphylos uva-ursi
Betula nana
Ectomycorrhizal Host species
36 new records for Scotland (28 UK)
23 taxa are undescribed - new to science
257 ECM taxa recorded
80 taxa appear restricted to the habitat
Alpine Ectomycorrhizal Fungi
Highly diverse, but poorly recorded
communities, restricted to a rapidly
declining habitat in Scotland.
Emily Carroll
Current Projects at Hutton
• Tree Health and Plant Biosecurity III
• UK Nursery testing
• Environmental diversity
• Baseline testing natural ecosystems
• Aids interception decisions
• Watershed sampling (WP1.3)
• Targeted sampling of catchment
WP1.3.3 Monitoring ECN sites
Environmental monitoring
THAPBI II nursery sampling
• 3 sites at 5-6 locations covering a range of habitats
• Additional key sites with help of Jenny Park (SNH)
Example 1
Hänfling et al., 2016 Environmental DNA metabarcoding of lake fish communities reflects long-term data from established survey methods. Molecular Ecology 25 3101-3119
• eDNA (14 species) better
than conventional gill-
netting (4 species)
• Interpretation challenges
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