Legal issues, logistics, data quality and acquisition, and collection/preservation methods with regards to field collecting.

1. Field Collecting for DNA Barcoding
Sarah Adamowicz & Alex Borisenko
Biodiversity Institute of Ontario & Dept. Integrative Biology
University of Guelph

2. Field Collecting: Considerations for DNA Barcoding
1- Permits
2- Collection and preservation
3- Data capture
4- Labeling
5- Plate thinking
6- Sampling effort

3. 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

4. Making Collections DNA-friendly: Preservation
Non-chemical preservation:
•Freezing – ideal, but expensive and logistically difficult
•Drying – good, but sensitive to storage environment
Chemical preservation (fluid fixation):
•Ethanol – good, common, but has issues
•DMSO, EDTA, SDS – good for DNA, but not morphology
NB! Do not change from one fixative to another!
All methods are sensitive to a wide range of factors:
•Quality of fixative
•Fixation procedure
•Storage conditions
•Nature and quality of tissue

5. 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)

6. Collecting and Preserving Specimens:
Summary of the Most Common Methods
• Freezing
• Insect kill jars (e.g. cyanide)
• Pinning
• Fluid: ethanol (remote locations
only if necessary: polypropylene
glycol with rapid transfer to
ethanol); exchange ethanol

7. Databasing and Labeling
• Capture information fresh
• Think plates from the
beginning
• Think high-throughput.

9. Pre-Lab Stages: Challenges
Different collections have different standards and traditions…
Transforming the diversity ...major
of collection management
approaches into standard
lab-compliant format... ? logistical
challenge!

10. Scaling Up: Transition to 96-well Sample Arrays
Single tube approach…
NOT SCALABLE!
Lab operates in a 96-
well plate format
Requires compatible
front-end solutions

11. Scaling Up: Specimen arraying
BIO collection: shifted arraying to specimen stage
Facilitates other front-end and curation stages:
•Imaging
•Tissue sampling
•Databasing
•Labelling

12. Key Stages of Front-end Processing: Summary
Transform collection specimens into
lab-ready arrays of tissue samples.
Specimen Data Specimen Tissue
arraying collection imaging sampling
NB! Do not include specimen collection, preparation and curation

13. Logistical Challenge: Specimen Arraying and Lots
Barcoding – Specimen-based Lot-based sampling
One specimen
One tissue sample
One data record
One DNA barcode Multiple specimens per lot
No easy solution, but there are ways to simplify sorting

14. 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.

15. 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

16. List of Label Assignments – Churchill 2010
Lots (L#10PROBE-0001…) Specimens (10PROBE-00001…)
Hannah & N/A 1500 (10PROBE-00001 – 10PROBE-01500)
Masha
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 1000 (L#10PROBE-7001 – L#10PROBE-8000) 10000 (10PROBE-18501 – 10PROBE-28500)
Ecology
Course
Extras

17. Sample ID = Plate Number + Well Locator
BIOUG0001-A01
BIOUG0001-A02
.
. BIO
.
BIOUG0001-H11
Can use “Field ID” and “Museum ID” columns for
other Specimen IDs needed. I use the “Field ID”
column for the lot number.

18. • 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.

19. 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

20. 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

21. Experience plays an important role in sampling
Amateur Expert
Example of Muscidae
- Jinjing Wang, Diptera of Churchill