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DNA Barcoding: Introduction to Front-End Processing of Biological Specimens Merging natural history with modern analytical workflows… Alex Borisenko, Biodiversity Institute of Ontario

The DNA Barcoding Workflow 3 Components: Collections, Molecular & Informatics Specimen Collection Data BOLD Systems Tissue Sample Photograph Web-Accessible Data and Extract DNA PCR Amplify Sequence DNA Barcodes

Front-end Processing: The Challenge Molecular approaches are scaling up... Collection processing is becoming the Why is this an issue? bottleneck and can impede large-scale DNA barcoding projects

Pre-Lab Processing: The Challenge Different collections have different standards and traditions… Search for ? Transforming the diversity is a major compromise of collection management logistical solutions approaches into standard challenge! lab-compliant format... Help collections with their needs Ensure lab-friendly format Project utility in non- barcoding applications

Logistical Challenge: Lots and Specimens Barcoding – Specimen-based Lot-based Sampling One specimen One tissue sample One data record Multiple specimens per lot One DNA barcode sequence Individual specimen tracking with data links to original lot

Logistical Challenge: Lots and Specimens Container with multiple individuals resulting from the same collecting event Lot Collection voucher representing a single Specimen biological individual

Logistical Challenge: Traditional Collection Management A Typical Collection Management Workflow: •Treats characteristics such as „lot‟ or „specimen‟ as storage unit attributes • Does not establish differential pathways for different types of storage units • Does not set a framework for fast processing of specimen aggregates Attribute: Lot (multiple unaccounted specimens; not suitable for routine barcoding) Storage unit Attribute: Specimen (represents biological individual; common source for DNA barcodes) Attribute: Fragment (Skin, skull, tissue, etc.) (represents part of biological individual)

Logistical Challenge: Scaling Up Molecular Analyses Single sample approach… NOT SCALABLE! Core labs operate in 96-well plate format Requires compatible front-end solutions:

The Solution: Develop Front-end Buffer Zone! PRE-LAB PROCESSING CORE LAB PROCESSES Arrayed plate Tissue lysis Arrayed tissue Tissue sampling DNA /subsampling extraction Unarrayed PCR gel check samples Arraying Seq. cleanup Cycle seq. Label data Databasing Imaging Preparation Whole vouchers Sequencing

Interfacing Between Collection and Lab Collection Management System Imaging Samples Data Images LIMS Core Lab Logistical independence of operations

Barcode Compliance: Specimen Requirements Samples are destined for analysis in a core sequencing facility Data are destined for centralized online repository (BOLD Systems) • Proper vouchering technique to preserve diagnostic morphology – very taxon-specific • Specimen numbering convention – to link the samples with their corresponding specimen records • Samples need to be preserved in a DNA-friendly fashion • The nature and quantity of samples has to be compatible with standard robotic DNA extraction protocols

Data Collection: BOLD Requirements

Data Collection: BOLD SpecimenData Spreadsheet MS Excel-based. Submitted by e-mail to BOLD team. 4 pages:

Specimen Imaging: BOLD Image Submission Protocol Imaging is important! Retention of an „electronic voucher‟

Barcode Compliance: Numbering Convention Darwin Core Triplet REPOSITORY: COLLECTION: catalog number Sample ID Field Number Catalog Number Collection Code Institution Storing MUS COL-000000 Field # or LOT # Museum number Collection Repository name NOTE1: Multiple barcode sequences for the same specimens are redundant. NOTE2: Catalog number must relate to biological individual (NOT to the lot #!) Registry of Biological Repositories Institutional Acronyms and Collections Codes www.biorepositories.org If your collection is not registered, please register!

Barcode Compliance: Voucher Archiving Retain reference to barcode on specimen! Barcode of Life Affix DNA voucher specimen „barcode Sample ID: MUS SP-00123 label‟ BOLD ID: CODE123-09 Barcode Label • Build virtual links to barcode vouchers • Tag vouchers with barcode information

Barcode Compliance: Provenance Information Possible data sources: Digital (ideal): • Collection database • Field data entry spreadsheets Data conversion required Analog (not preferred): • Collection archive • Field documentation • Specimen labels Data digitization required

Barcode Compliance: Data Management Other ways to improve data quality... • Standardize metadata fields for provenance information that is being collected (compatibility with Darwin Core v.2) • Taxonomic framework: global checklists, standards for interim taxonomy, resolution of nomenclatural disputes • Geographic framework: standard administrative divisions, agree on transliterations – ISO country and province codes; decimal coordinates, standard datum (WGS84) • Agree on taxon-specific extra information to be collected (relevant to taxonomy or ecological interactions)

DNA-friendly Sampling: Tissue Source Main features of a barcode-friendly tissue source: • Mitochondria-rich • Low enzymatic activity • Easy lysis (but not autolysis) • Low risk of foreign contaminants  DNA-friendly sources: • Arthropod legs • Muscle • Brain • Gonad (not in insects) Discouraged sources: • Guts • Liver & internal organs Avoid cross-contamination!

DNA-friendly Collecting: Specimen Fixation DNA preservation (or degradation) starts during collection (killing method, exposure to elements, etc.) Chris Meyer: “Get rid of water & shut down nucleases” DNA-friendly killing/fixation methods:  • Non-chemical methods (Freezing) • Ethanol (aquatic, pitfalls and malaise traps) • Chloroform, Cyanide, Ammonia (insects) • Isoflurane, carbon dioxide (vertebrates) DISCOURAGED killing/fixation 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 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: • Nature and quality of tissue • Quality of fixative/preservative • Fixation procedure • Storage conditions

Making Collections DNA-friendly: Other Factors Example: Ethanol Specimen • 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 Specimen • Drying conditions • Pretreatment (skin tanning, insect relaxing) • Ambient humidity • Storage temperature • Exposure to sunlight • Fumigants and preservatives used (PDB, arsenic)

Specimen Transactions and Data Policies The need for streamlined and efficient tracking

Specimen Transactions and Data Policies Clear material transfer protocols (BMTA) BMTA and Transparent data release policy • How will the materials be stored and analyzed? • How will molecular data and collaterals be released? Balance ownership rights and community services

Front-end Processing: Workshop Highlights Transform collection specimens into lab-ready arrays of tissue samples. Specimen Specimen Data Tissue arraying imaging collection sampling

Front-end Processing – from the field to the lab • Data Collection • Imaging Procedures • Sampling Procedures • Sampling Kits

Specimen/Sample Arraying: The Concept So, what is that array thing anyway? 12 × 8 format D04 Specimen aggregate matching plate map 95 samples + 1 control

Specimen/Sample Arraying: Examples Subsampling Direct sampling • Databasing Enables batch tracking • Imaging through front-end and • Tissue sampling curation stages: • Labelling

Sampling Kits: to Streamline Sample Submission... Dispatched by core analytical facilities... CD with templates Sampling medium Sampling instructions for data entry Microplate BMTA & Data Policy Agreement (iBOL) Tube Rack Plant Tube Rack

Sampling Kits: What‟s included? Plate color coding Sampling Kit Data Package: • SpecimenData.xls • ImageData.xls DNA PCR • CCDB-0000_Record.xls Plate map – critical component Tissue Cap strips

Data Collection: Digitize data right in the field! Use custom spreadsheet templates for data tracking and field collection management • MS Excel Platform – familiar interface • Built-in formulas – data conversion and parsing • Built-in macros – automation of data transfer • Tracks the entire chain from field collecting to BOLD submission and museum deposition (Darwin Core)