Leighton Pritchard & Peter Thorpe Sarah Green Tree Health and - - PowerPoint PPT Presentation

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Leighton Pritchard & Peter Thorpe Sarah Green Tree Health and - - PowerPoint PPT Presentation

Jul 08, 2023 221 likes •468 views

Use of Technology in the Natural Environment SNH Sharing Good Practice day June 7 2017 eDNA and Plant Pathogen Metabarcoding David Cooke Leighton Pritchard & Peter Thorpe Sarah Green Tree Health and Plant Biosecurity initiative

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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

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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

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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

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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
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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
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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

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Case Study - Phytophthora

  • 167 species – destructive plant pathogens
  • 16 species on UK plant health risk register
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Dave Rizzo Youtube.com

California Oak Mortality Task Force

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Phytophthora zoospore detection

Sampling water

  • Irrigation water
  • Water flooded through roots
  • f pot-grown plants
  • Rivers

Filtration

  • Cellulose acetate filters
  • DNA extraction
  • PCR - Phytophthora genus

specific primers

  • Sequencing (high or low

throughput)

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  • 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…

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Some species abundant all year

Quantitative when considering frequency of occurrence between samples

Winter months

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  • P. xcambivora lower frequency all year

Winter months

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Unknown clade 4 species – summer only

Winter months

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Herbaceous downy mildew (nettle) summer only

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Mycorrhizal Fungi in Montane Heaths

Dr Andy Taylor – James Hutton Institute, Aberdeen

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Arctostaphylos alpinus Salix herbecea Arctostaphylos uva-ursi Betula nana

Ectomycorrhizal Host species

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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

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Alpine Ectomycorrhizal Fungi

Highly diverse, but poorly recorded communities, restricted to a rapidly declining habitat in Scotland.

Emily Carroll

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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
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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)

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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