Field Collecting for DNA Barcoding

Sarah Adamowicz & Alex Borisenko

Biodiversity Institute of Ontario & Dept. Integrative Biology

University of Guelph

Field Collecting: Considerations for DNA Barcoding

1- Permits

2- Collection and preservation

3- Data capture

4- Labeling

5- Plate thinking

6- Sampling effort

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

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

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)

• 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

• Capture information fresh

• Think plates from the beginning

• Think high-throughput.

Databasing and Labeling

...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…

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!

Scaling Up: Specimen arraying

BIO collection: shifted arraying to specimen stage

Facilitates other front-end and curation stages:

•Imaging

•Tissue sampling

•Databasing

•Labelling

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

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

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.

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

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

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.

• 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.

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

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

Experience plays an important role in sampling

Amateur Expert

Example of Muscidae

- Jinjing Wang, Diptera of Churchill