DNA Barcoding: An Emerging Global Standard for Species Identification

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DNA Barcoding An Emerging Global Standard for
Species Identification

• Consortium for the Barcode of Life
• National Museum of Natural History
• Smithsonian Institution
• http//www.barcoding.si.edu
• 202/633-0808 fax [phone]

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A DNA barcode is a short gene sequence taken
from standardized portions of the genome, used
to identify species
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Characteristics of Barcode Regions

• Flanked by conserved regions
• Easy to amplify
• Low intraspecies variability
• Discontinuous variation between species
• Long enough to work in all groups
• Short enough for single reads

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The Mitochondrial Genome

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Using DNA Barcodes

• Establish reference library of barcodes from
  identified voucher specimens
• If necessary, revise species limits
• Then
• Identify unknowns by searching against reference
  sequences
• Look for matches (mismatches) against library on
  a chip
• Before long Analyze relative abundance in
  multi-species samples

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Analytical chain

• Databasing
• Labeling
• Imaging
• Tissue sampling
• DNA extraction
• PCR
• PCR check
• Sequencing reaction
• Sequencing cleanup
• Sequencing
• Trace editing submission

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BoLD Data System

• Developed/hosted by Univ. Guelph
• Workbench for most barcode projects
• Laboratory Information Management System (LIMS)
  for assembling data
• Management and Analysis System
• Identification system for matching unknowns to
  reference records
• Uploading to GenBank

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Methods
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Barcode of Life Database
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Analytical chain

• Databasing
• Labeling
• Imaging
• Tissue sampling
• DNA extraction
• PCR
• PCR check
• Sequencing reaction
• Sequencing cleanup
• Sequencing
• Trace editing submission

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Current Norm High throughput
Large capacity PCR and sequencing reactions
ABI 3100 capillary automated sequencer
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Cost of Reagents and Disposables

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Producing Barcode Data 2008 Faster, more
portable Hundreds of samples per hour
Integrated DNA microchips
Table-top microfluidic systems
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Producing Barcode Data 2010?Barcode data
anywhere, instantly

• Data in seconds to minutes
• Pennies per sample
• Link to reference database
• A taxonomic GPS
• Usable by non-specialists

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Methods
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Uses of DNA Barcodes

• Applied tool for identifying regulated species
• Disease vectors, agricultural pests, invasives
• Environmental indicators, protected species
• Using minimal samples, damaged specimens, gut
  contents, droppings
• Research tool for improving species-level
  taxonomy
• Associating all life history stages, genders
• Testing species boundaries, finding new variants
• Triage tool for flagging potential new species
• Undescribed and cryptic species

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Uses of DNA Barcodes

• Applied tool for identifying regulated species
• Disease vectors, agricultural pests, invasives
• Environmental indicators, protected species
• Using minimal samples, damaged specimens, gut
  contents, droppings
• Research tool for improving species-level
  taxonomy
• Associating all life history stages, genders
• Testing species boundaries, finding new variants
• Triage tool for flagging potential new species
• Undescribed and cryptic species

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Associating Life Stages, Processed Parts,
Dimorphic Genders
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Steatogenini until the early 90s
Hypopygus lepturus Hoedeman 1962
Steatogenys elegans Steatogenys duidae
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Color patterns in Hypopygus
Nijssen Isbrüker 1972
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Steatogenini during the 90s
Hypopygus lepturus Hoedeman 1962
Hypopygus neblinae Mago-Leccia 1994
Steatogenys
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Steatogenini during the 90s / today
Hypopygus lepturus Hoedeman 1962
Hypopygus neblinae Mago-Leccia 1994
Stegostenopos Triques 1997
Steatogenys
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R. Bernhard, 2004
8a
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RAG 1 MP/ML/Dist
Stegostenopus
Hypopygus neblinae
A
H. lepturus
C
D
Steatogenys
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12S16S Strict of ML/MP/Dist
Stegostenopus
H. neblinae
A
C
H. lepturus
D
E
Steatogenys
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D-loop MP/ML/Dist
H. lepturus
D
E
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COI - BARCODE MP
H. lepturus
Eigenmannia sp.
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Uses of DNA Barcodes

• Applied tool for identifying regulated species
• Disease vectors, agricultural pests, invasives
• Environmental indicators, protected species
• Using minimal samples, damaged specimens, gut
  contents, droppings
• Research tool for improving species-level
  taxonomy
• Associating all life history stages, genders
• Testing species boundaries, finding new variants
• Triage tool for flagging potential new species
• Undescribed and cryptic species

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Wider Impacts of Barcoding 2008

• Catalyzing interoperability of databases
• Barcode data standards link sequences, specimens,
  species names and publications
• Improving the information infrastructure
• Digital library initiative in taxonomy
• Renewing the mission of museums
• DNA recovery from formalin-fixed specimens
• Promoting the growth of DNA banks
• Expanding analytical toolbox for taxonomy

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What DNA Barcoding is NOT

• Barcoding is not DNA taxonomy no single gene (or
  character) is adequate
• Barcoding is not Tree of Life barcode clusters
  are not phylogenetic trees
• Barcoding is not just COI standardizing on one
  region has benefits and limits
• Molecules in taxonomy is not new but large-scale
  and standardization are new
• Barcoding can help to create a 21st century
  research environment for taxonomy

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What DNA Barcoding is NOT

• Barcoding is not DNA taxonomy no single gene (or
  character) is adequate
• Barcoding is not Tree of Life barcode clusters
  are not phylogenetic trees
• Barcoding is not just COI standardizing on one
  region has benefits and limits
• Molecules in taxonomy is not new but large-scale
  and standardization are new
• BUTBarcoding can help to create a 21st century
  research environment for taxonomy

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Consortium for the Barcode of Life (CBOL)

• First barcoding publications in 2002
• Cold Spring Harbor planning workshops in 2003
• Sloan Foundation grant, launch in May 2004
• Secretariat opens at Smithsonian, September 2004
• First international conference February 2005
• Now an international affiliation of
• 130 Members Orgs, 40 countries, 6 continents
• Natural history museums, biodiversity
  organizations
• Users e.g., government agencies
• Private sector biotech companies, database
  providers

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CBOL Member Organizations June 2006 120 Member
Organizations, 40 countries
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CBOLs Working Groups

• Database Designing/constructing the Barcode
  Section of GenBank
• DNA Protocols for formalin-fixed and old museum
  specimens Producing LIMS for dissemination
• Data Analysis Beyond phenetic methods
  population genetics perspective
• Plants Identify gene region(s) for barcoding

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Infrastructure of TaxonomyFragmented,
Disconnected

• Collections and databases of specimens
• Compilations of taxonomic names
• Data repositories (characters, gene sequences,
  images, trees)
• Monographs
• Floristic and faunistic surveys/inventories
• Revisions
• The (undigitized) Taxonomic Literature

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Barcode Records in INSDC

• Consensus results of Front Royal meeting
• GBIF ? ITIS ? GRIN
• NBII ? Species2000 ? IPNI
• ICZN ? ZooRecord ? OBIS
• Structured link to voucher specimen
• Species name selected from authority
• Online access to metadata
• Trace files and quality scores
• Minimum sequence length

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BARCODE records in GenBank
Voucher Specimen
Species Name
Specimen Metadata
GeoreferenceHabitatCharacter setsImagesBehavio
rOther genes
Indices - Catalog of Life - GBIF/ECAT Nomenclato
rs - Zoo Record - IPNI NameBank Publication
links - New species
Barcode Sequence
Trace files
Primers
Other Databases
Literature(link to content or citation)
PhylogeneticPopn GeneticsEcological
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Digitizing Taxonomic Literature

• CBOLs catalytic efforts
• Library-Laboratory meeting in London on
  electronic access to taxonomic literature
• Led to formation of Biodiversity Heritage Library
  initiative
• Proactive steps with PubMed to add taxonomic
  journals to online abstracts
• Aggressive negotiation with publishers of
  barcoding papers

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CBOLs Working Groups

• Database Designing/constructing the Barcode
  Section of GenBank
• DNA Protocols for formalin-fixed and old museum
  specimens Producing LIMS for dissemination
• Data Analysis Beyond phenetic methods
  population genetics perspective
• Plants Identify gene region(s) for barcoding

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The Barcode Assembly Line 2006
Freshly collected specimens
Young museum specimens
Frozen tissue
DNA Barcode Data
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The Barcode Assembly Line 2008Opening the
museum treasure-trove
Freshly collected specimens
Formalin-fixed specimens
Older museum specimens
Young museum specimens
Frozen tissue
DNA Barcode Data
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CBOL Formalin Workshop

• Literature survey of DNA recovery protocols from
  formalin-fixed specimens
• Solicited proposal from National Research Council
• May 8-9 workshop in Washington
• Chemists, biochemists, biophysicists, biomedical
  researchers
• Create a new research agenda

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CBOLs Working Groups

• Database Designing/constructing the Barcode
  Section of GenBank
• DNA Protocols for formalin-fixed and old museum
  specimens Producing LIMS for dissemination
• Data Analysis Beyond phenetic methods
  population genetics perspective
• Plants Identify gene region(s) for barcoding

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Data analysis protocols in 2008 A Bigger, Better
Analytical Toolkit to handle the Barcode Data
Explosion

• Collaboration of statisticians, computer
  scientists, population geneticists
• Sampling issues
• Sample size versus confidence level
• Sample size in light of geography, gene flow
• Analytical tools and protocols
• Treatment of missing DNA site data
• Identification versus species delimitation
  (classification versus clustering)

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CBOLs Working Groups

• Database Designing/constructing the Barcode
  Section of GenBank
• DNA Protocols for formalin-fixed and old museum
  specimens Producing LIMS for dissemination
• Data Analysis Beyond phenetic methods
  population genetics perspective
• Plants Identify barcode gene region(s) for
  land plants

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Progress toward Plant Barcode

• Kress 2005 proposal for ITS and trnh-psbA
• Kew Garden receives Sloan/Moore Foundation
  support
• Phase 1 screens 100 genes across 50 sibling
  species pairs
• Phase 2 tests of matK, rpcoC1, rpoB, ndhJ, and
  accD
• Canadian proposal for rbcL
• CBOL protocols for approving barcode regions

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Current and Planned CBOL Barcoding Projects

• FishBOL and All Birds Initiatives
• Demonstrator Systems by 2008
• Tephritid fruit flies (agricultural pests)
• Mosquitoes (disease vectors)
• African Scale Insect Barcoding Initiative
  (planned at Cape Town Regional Meeting)
• Barcoding for Conservation Committee

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Launching CBOL Projects

• Assembling Steering Committee
• Users
• Taxonomists, collection curators
• Service providers (BoLD, analytical labs)
• Plan for scope, timetable, logistics
• Pilot tests of primers, PCR amplification
• Assemble pipeline of specimens to lab

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ABBI and FISH-BOL

• Global initiatives to create reference library
• Enable users to adopt barcode ID systems
• All-species barcode database will
• Strengthen specimen/species data
• Improve collections, tissue/DNA resources
• Attract users to barcoding for specimen IDs
• Regional Working Groups
• Small Steering Committee and CBOL

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Planned Outreach

• Regional meetings in
• Cape Town, South Africa, 7-8 April 2006, SANBI
• Nairobi, Kenya, 18-19 October 2006, NMK
• Sao Paolo, Brazil, February 2007, INPA
• Southern/SE Asia, mid-2007
• Second International Barcode Conference
• Southeast Asia (?), September 2007 (?)
• Support from CBOL, host governments and
  international development agencies

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Milestones for 2008
2007
2008
2006
Q1
Q2
Q3
Q4
Q1
Q2
Q3
Q4
Q1
Q2
200K records
500K records
100K records
Database
Formalin Study
Advanced Lab Protocols
DNA WG
Development of Consensus Plant Barcode Region
Plant WG
International Conference
Demonstrator System Launched
Data Analysis Protocols and S/W
Data Analysis WG
BoLI Data Portal Launched
Extended DB Interoperability
Data Standards
Database WG
Campaigns
Regional Groups Operational
First Data Releases
10K birds30K fish