Field Collecting for DNA Barcoding Sarah Adamowicz & Alex BorisenkoBiodiversity 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 CollectionDNA 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 solutionsNB! Ensure timely preservation adequate for material
Making Collections DNA-friendly: PreservationNon-chemical preservation:•Freezing – ideal, but expensive and logistically difficult•Drying – good, but sensitive to storage environmentChemical 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
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)
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
Databasing and Labeling• Capture information fresh• Think plates from thebeginning• Think high-throughput.
Pre-Lab Stages: Challenges Different collections have different standards and traditions…Transforming the diversity ...majorof collection managementapproaches into standardlab-compliant format... ? logistical challenge!
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
Scaling Up: Specimen arrayingBIO collection: shifted arraying to specimen stageFacilitates 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 Data Specimen Tissue arraying collection imaging samplingNB! Do not include specimen collection, preparation and curation
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
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 andspecimens – quota to each researcher• Consecutive lot numbers andspecimen IDs, e.g.L#09PROBE-0001Churchill, MB, Can, July 14-31, 200909PROBE-00001Churchill, MB, Can, July 14-31, 2009• Spreadsheet that outputs labelsand outputs straight to BOLD format
Sample ID = Plate Number + Well LocatorBIOUG0001-A01BIOUG0001-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.
• Jinjing Wang• Diptera of Churchill.• Collected for 3 months• Prepared 9,000specimens for barcodingin 6 months (sorting,family IDs, databasing,labeling, arraying,photographing, tissuesampling, data upload toBOLD)• Molecular work completein 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
Conductingbiodiversity surveys:Detecting undersampling in the Tipulidae (crane flies) of Churchill After 2007, 24 putative species and numerous singletonsAfter expert collection in 2008, 42 species
Experience plays an important role in sampling Amateur Expert Example of Muscidae - Jinjing Wang, Diptera of Churchill