Download - Dr Sarah Adamowicz - Field collections
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Field Collecting for DNA Barcoding
Sarah Adamowicz & Alex Borisenko
Biodiversity Institute of Ontario & Dept. Integrative Biology
University of Guelph
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Field Collecting: Considerations for DNA Barcoding
1- Permits
2- Collection and preservation
3- Data capture
4- Labeling
5- Plate thinking
6- Sampling effort
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Making Collections DNA-friendly: Specimen Collection
DNA preservation (or degradation) starts during collection
(killing method, exposure to elements, etc.)
DNA-friendly killing methods:
•Non-chemical methods (Freezing)
•Ethanol (aquatic, pitfalls and malaise traps)
•Chloroform, Cyanide, Ammonia (insects)
•Isoflurane, carbon dioxide (vertebrates)
DISCOURAGED killing methods:
•Formalin (marine)
•Ethyl acetate (insects)
•Diluted propylene glycol (malaise traps, pitfalls)
•Most histological solutions
NB! Ensure timely preservation adequate for material
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Making Collections DNA-friendly: Preservation
Non-chemical preservation:
•Freezing – ideal, but expensive and logistically difficult
•Drying – good, but sensitive to storage environment
NB! Do not change from one fixative to another!
Chemical preservation (fluid fixation):
•Ethanol – good, common, but has issues
•DMSO, EDTA, SDS – good for DNA, but not morphology
All methods are sensitive to a wide range of factors:
•Quality of fixative
•Fixation procedure
•Storage conditions
•Nature and quality of tissue
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Making Collections DNA-friendly: Contributing Factors
Example: Ethanol fixation
•Quality (e.g., acidity and additives)
•Reagent concentration (water content)
•Tissue/Ethanol volume ratio
•Relative surface area of sample
•Storage temperature
•Exposure to light
•Fixative evaporation
Example: dry sample
•Drying conditions
•Pretreatment (skin tanning, insect relaxing)
•Ambient humidity
•Storage temperature
•Exposure to sunlight
•Fumigants and preservatives used (PDB, arsenic)
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• Freezing
• Insect kill jars (e.g. cyanide)
• Pinning
• Fluid: ethanol (remote locations only if necessary: polypropylene glycol with rapid transfer to ethanol); exchange ethanol
Collecting and Preserving Specimens: Summary of the Most Common Methods
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• Capture information fresh
• Think plates from the beginning
• Think high-throughput.
Databasing and Labeling
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...major
logistical
challenge!
Pre-Lab Stages: Challenges
?Transforming the diversity
of collection management
approaches into standard
lab-compliant format...
Different collections have different standards and traditions…
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Scaling Up: Transition to 96-well Sample Arrays
Single tube approach…
Lab operates in a 96-
well plate format
Requires compatible
front-end solutions
NOT SCALABLE!
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Scaling Up: Specimen arraying
BIO collection: shifted arraying to specimen stage
Facilitates other front-end and curation stages:
•Imaging
•Tissue sampling
•Databasing
•Labelling
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Key Stages of Front-end Processing: Summary
Transform collection specimens into
lab-ready arrays of tissue samples.
Specimen
arraying
Specimen
imagingData
collection
Tissue
sampling
NB! Do not include specimen collection, preparation and curation
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Barcoding – Specimen-based
One specimen
One tissue sample
One data record
One DNA barcode
Lot-based sampling
Multiple specimens per lot
No easy solution, but there are ways to simplify sorting
Logistical Challenge: Specimen Arraying and Lots
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Custom Solutions for Specimen Databasing in the Field
Features:
• Simplicity
• Data validation
• Label printing
• BOLD Data conversion
• Taxonomic curation
Alex Borisenko, Curator of Zoological Collections, Biodiversity Institute of
Ontario: Multi-page electronic spreadsheet – full autonomy.
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Field Labels & Permanent Labels
• Standardized labels for both lots and specimens – quota to each researcher
• Consecutive lot numbers and specimen IDs, e.g.
L#09PROBE-0001
Churchill, MB, Can, July 14-31, 2009
09PROBE-00001
Churchill, MB, Can, July 14-31, 2009
• Spreadsheet that outputs labels
and outputs straight to BOLD format
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Lots (L#10PROBE-0001…) Specimens (10PROBE-00001…)
Hannah &
Masha
N/A 1500 (10PROBE-00001 – 10PROBE-01500)
Brandon 1000 (L#10PROBE-0001 – L#10PROBE-1000) 2000 (10PROBE-01501 – 10PROBE-03500)
Liz 1000 (L#10PROBE-01001 – L#10PROBE-2000) 2000 (10PROBE-03501 – 10PROBE-05500)
Emily 1000 (L#10PROBE-2001 – L#10PROBE-3000) 2000 (10PROBE-05501 – 10PROBE-07500)
Jinjing 1000 (L#10PROBE-3001 – L#10PROBE-4000) 3000 (10PROBE-07501 – 10PROBE-10500)
Kara 1000 (L#10PROBE-4001 – L#10PROBE-5000) 2000 (10PROBE-10501 – 10PROBE-12500)
Monica 1000 (L#10PROBE-5001 – L#10PROBE-6000) 2000 (10PROBE-12501 – 10PROBE-14500)
Fatima 500 (L#10PROBE-6001 – L#10PROBE-6500) 2000 (10PROBE-14501 – 10PROBE-16500)
Vadim 500 (L#10PROBE-6501 – L#10PROBE-7000) 2000 (10PROBE-16501 – 10PROBE-18500)
Arctic
Ecology
Course
1000 (L#10PROBE-7001 – L#10PROBE-8000) 10000 (10PROBE-18501 – 10PROBE-28500)
Extras
List of Label Assignments – Churchill 2010
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BIO
BIOUG0001-A01
BIOUG0001-A02
.
.
.
BIOUG0001-H11
Sample ID = Plate Number + Well Locator
Can use “Field ID” and “Museum ID” columns for
other Specimen IDs needed. I use the “Field ID”
column for the lot number.
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• Jinjing Wang
• Diptera of Churchill.
• Collected for 3 months
• Prepared 9,000
specimens for barcoding
in 6 months (sorting,
family IDs, databasing,
labeling, arraying,
photographing, tissue
sampling, data upload to
BOLD)
• Molecular work complete
in 2 months.
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Field: Planning Sampling Effort
• What is “complete”? What is the goal?
• How do you know when you have reached the goal?
• accumulation curves
• non-parametric estimators of diversity (program EstimateS)
• checklists, if available, but with caution
• Importance of sampling multiple times
• Importance of expert collectors
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Conducting
biodiversity surveys:
Detecting undersampling
in the Tipulidae (crane
flies) of Churchill
After 2007, 24 putative
species and numerous
singletons
After expert collection in
2008, 42 species
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Experience plays an important role in sampling
Amateur Expert
Example of Muscidae
- Jinjing Wang, Diptera of Churchill
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