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Dr Sarah Adamowicz - Field collections
 

Dr Sarah Adamowicz - Field collections

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

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

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    Dr Sarah Adamowicz - Field collections Dr Sarah Adamowicz - Field collections Presentation Transcript

    • 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
    • List of Label Assignments – Churchill 2010 Lots (L#10PROBE-0001…) Specimens (10PROBE-00001…)Hannah & N/A 1500 (10PROBE-00001 – 10PROBE-01500)MashaBrandon 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)EcologyCourseExtras
    • 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