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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,
Merging natural history with modern analytical workflows…
DNA Barcoding: Introduction to Front-End Processing
Alex Borisenko, Biodiversity Institute of Ontario
The DNA Barcoding Workflow
BOLD Systems
Web-Accessible Data and DNA Barcodes Specimen Collection Data Tissue Sample Photograph PCR Amplify Sequence Extract DNA
3 Components: Collections, Molecular & Informatics
Front-end Processing: The Challenge
Why is this an issue?
Molecular approaches are scaling up... Collection processing is becoming the bottleneck and can impede large-scale DNA barcoding projects
is a major logistical challenge!
Transforming the diversity
approaches into standard lab-compliant format...
Different collections have different standards and traditions…
Search for compromise solutions
Help collections with their needs Ensure lab-friendly format Project utility in non- barcoding applications
Pre-Lab Processing: The Challenge
Lot-based Sampling
Multiple specimens per lot Individual specimen tracking with data links to original lot
Barcoding – Specimen-based
One specimen One tissue sample One data record One DNA barcode sequence
Logistical Challenge: Lots and Specimens
Lot
Container with multiple individuals resulting from the same collecting event
Specimen
Collection voucher representing a single biological individual
Logistical Challenge: Lots and Specimens
Logistical Challenge: Traditional Collection Management
Attribute: Lot (multiple unaccounted specimens; not suitable for routine barcoding) Attribute: Specimen (represents biological individual; common source for DNA barcodes) Attribute: Fragment (Skin, skull, tissue, etc.) (represents part of biological individual)
Storage unit
A Typical Collection Management Workflow:
Single sample approach…
Core labs operate in 96-well plate format
Requires compatible front-end solutions:
NOT SCALABLE! Logistical Challenge: Scaling Up Molecular Analyses
The Solution: Develop Front-end Buffer Zone!
PRE-LAB PROCESSING CORE LAB PROCESSES
DNA extraction PCR Cycle seq. gel check
Sequencing Tissue lysis
Arrayed plate
Imaging Tissue sampling /subsampling Preparation Databasing Arraying Whole vouchers Label data Unarrayed samples Arrayed tissue
Interfacing Between Collection and Lab
Core Lab Imaging LIMS Collection Management System
Samples
Data Images
Logistical independence of operations
Samples are destined for analysis in a core sequencing facility
morphology – very taxon-specific
with their corresponding specimen records
with standard robotic DNA extraction protocols
Barcode Compliance: Specimen Requirements
Data are destined for centralized online repository (BOLD Systems)
Data Collection: BOLD Requirements
MS Excel-based. Submitted by e-mail to BOLD team. 4 pages:
Data Collection: BOLD SpecimenData Spreadsheet
Specimen Imaging: BOLD Image Submission Protocol
Imaging is important! Retention of an „electronic voucher‟
Darwin Core Triplet REPOSITORY: COLLECTION: catalog number
NOTE2: Catalog number must relate to biological individual (NOT to the lot #!)
Sample ID Field Number Catalog Number Collection Code Institution Storing MUS COL-000000 Field # or LOT # Museum number Collection Repository name
www.biorepositories.org
NOTE1: Multiple barcode sequences for the same specimens are redundant.
Registry of Biological Repositories
Institutional Acronyms and Collections Codes
Barcode Compliance: Numbering Convention
If your collection is not registered, please register!
Retain reference to barcode on specimen!
Barcode of Life
DNA voucher specimen Sample ID: MUS SP-00123 BOLD ID: CODE123-09
Barcode Label
Barcode Compliance: Voucher Archiving
Affix „barcode label‟
Digital (ideal):
Data conversion required
Possible data sources:
Analog (not preferred):
Data digitization required
Barcode Compliance: Provenance Information
Barcode Compliance: Data Management
Other ways to improve data quality...
that is being collected (compatibility with Darwin Core v.2)
interim taxonomy, resolution of nomenclatural disputes
agree on transliterations – ISO country and province codes; decimal coordinates, standard datum (WGS84)
(relevant to taxonomy or ecological interactions)
DNA-friendly sources:
Discouraged sources:
Main features of a barcode-friendly tissue source:
Avoid cross-contamination!
DNA-friendly Sampling: Tissue Source
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:
DISCOURAGED killing/fixation methods:
NB! Ensure timely preservation adequate for material
DNA-friendly Collecting: Specimen Fixation
Non-chemical preservation:
NB! Do not change from one fixative to another! Chemical preservation (fluid fixation):
All methods are sensitive to a wide range of factors:
Making Collections DNA-friendly: Preservation
Example: Ethanol Specimen
Example: Dry Specimen
Making Collections DNA-friendly: Other Factors
Specimen Transactions and Data Policies
The need for streamlined and efficient tracking
Specimen Transactions and Data Policies BMTA
Clear material transfer protocols (BMTA) and Transparent data release policy
Balance ownership rights and community services
Transform collection specimens into lab-ready arrays of tissue samples.
Specimen arraying Specimen imaging Data collection Tissue sampling
Front-end Processing: Workshop Highlights
Front-end Processing – from the field to the lab
So, what is that array thing anyway?
Specimen aggregate matching plate map
12×8 format
95 samples + 1 control
Specimen/Sample Arraying: The Concept
D04
Enables batch tracking through front-end and curation stages:
Specimen/Sample Arraying: Examples
Subsampling Direct sampling
Sampling Kits: to Streamline Sample Submission...
Sampling instructions Sampling medium
Microplate Plant Tube Rack Tube Rack
BMTA & Data Policy Agreement (iBOL) CD with templates for data entry Dispatched by core analytical facilities...
Sampling Kits: What‟s included?
Plate color coding
DNA PCR
Tissue Sampling Kit Data Package:
Plate map–critical component Cap strips
submission and museum deposition (Darwin Core)
Use custom spreadsheet templates for data tracking and field collection management
Data Collection: Digitize data right in the field!
Expedition or collecting trip
A large-scale collecting effort spanning a certain geographic area May or may not target specific groups or habitats Diverse suite of collecting methods
A small-scale collecting effort targeting a certain habitat or focal group
Collecting Event
May or may not be successful
Specimen Imaging: Photography Setup
It's not about the gear!
High image quality can be achieved with moderate investments – be creative!
See video example…
Specimen Imaging: Framing & Orientation
Framing:
possible!
specimen
Orientation:
diagnostic characters
batch of specimens
ImageData submission sheet
Specimen Imaging: Background
Black… Or white?
Colour backgrounds are impractical.
Specimen Imaging: Background
Practical Examples: Tool Sterilization
Flame sterilization (DNA-poor tissue)
ELIMINase sterilization (DNA-rich tissue)
NB! Choose the right method!
Conclusion: The “Barcoding Pyramid”
End user
High-tech stages
are critically dependent
sourcing Specimen sourcing is dependent
biodiversity community