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Species Identification, Species Identification, Regulatory Agencies and Regulatory Agencies and
DNA BarcodingDNA Barcoding
David E. Schindel, Executive SecretaryDavid E. Schindel, Executive SecretaryNational Museum of Natural HistoryNational Museum of Natural History
Smithsonian InstitutionSmithsonian Institution
[email protected];; http://www.barcoding.si.edu202/633-0812; fax 202/633-2938202/633-0812; fax 202/633-2938
Genomics
Subgenomics
Current SystematicStudies
Microbes - 16SPlants - RBCLAnimals - COI
Species Identification MattersSpecies Identification MattersBasic research on evolution, ecologyBasic research on evolution, ecologyEndangered/protected species Endangered/protected species Agricultural pests/beneficial speciesAgricultural pests/beneficial speciesDisease vectors/pathogensDisease vectors/pathogensInvasive species (e.g., in ballast water)Invasive species (e.g., in ballast water)Environmental quality indicatorsEnvironmental quality indicatorsManaging for sustainable harvestingManaging for sustainable harvestingConsumer protection, ensuring food qualityConsumer protection, ensuring food qualityFidelity of seedbanks, culture collectionsFidelity of seedbanks, culture collections
5
Specimens
Characters
Distributions of Character Variation
Taxonomic Decision-Making
Socioeconomic Decisions
Specimens
Concerns/ Regulations
The Practice of Taxonomy
Taxonom
ists
The Uses of Taxonomy
Taxonomic ProcessesTaxonomic Processes
Specimens
Taxon Concepts
Characters
Formal Taxa
Growth of Biodiversity DatabasesGrowth of Biodiversity Databases
Journal Publication
Species Name
Voucher Specimen
Authority files of taxonomic
names
Museum databases of
associated data
Biodiversity Heritage Library
A DNA barcode is a A DNA barcode is a short gene sequence short gene sequence
taken from taken from standardized portions standardized portions
of the genome,of the genome, used to identify species used to identify species
An Internal ID System for All Animals
Typical Animal Cell
Mitochondrion
DNA
mtDNA
D-Loop
ND5
H-strand
ND4
ND4L
ND3COIII
L-strand
ND6
ND2
ND1
COII
Small ribosomal RNA
ATPase subunit 8
ATPase subunit 6
Cytochrome b
COICOI
The Mitochondrial Genome
Non-COI regions for other taxaNon-COI regions for other taxa
Land plants:Land plants:– Chloroplast Chloroplast matKmatK and and rbcLrbcL approved Nov 09 approved Nov 09– Non-coding plastid and nuclear regions being Non-coding plastid and nuclear regions being
exploredexplored
Fungi and protists:Fungi and protists:– CBOL Working Groups convenedCBOL Working Groups convened– Recommendations expected in 2010Recommendations expected in 2010
How Barcoding WorksHow Barcoding WorksFirst, build a barcode reference library:First, build a barcode reference library:– Well-identified specimenWell-identified specimen– Tissue subsampleTissue subsample– DNA extraction, PCR amplificationDNA extraction, PCR amplification– DNA sequencingDNA sequencing– Data submission to GenBankData submission to GenBank
Second, use it to identify unknowns:Second, use it to identify unknowns:– Any unidentified juvenile, adult, fragment, productAny unidentified juvenile, adult, fragment, product– Tissue sample, DNA, sequencingTissue sample, DNA, sequencing– Comparison with sequences in reference libraryComparison with sequences in reference library
Associating Life Stages, Processed Parts, Dimorphic Genders
How Barcoding is Done
From specimen to sequence to species
Voucher Specimen
DNA extraction CO1 gene DNA sequencing Trace file
Database of Barcode Records
Collecting
ND3
COIII
ND2
ND1
NBII, 25 February 2009NBII, 25 February 2009
GenBank, EMBL, and DDBJGenBank, EMBL, and DDBJGlobal, Open Access to Barcode DataGlobal, Open Access to Barcode Data
http://www.insdc.org/
Current Norm: High throughputLarge labs, hundreds of samples per day
ABI 3100 capillary
automated sequencer
Large capacity PCR and
sequencing reactions
Emerging Norm: Table-top LabsEmerging Norm: Table-top Labs Faster, more portable: Hundreds of samples per hour Faster, more portable: Hundreds of samples per hour
Integrated DNA microchips Table-top microfluidic systems
Producing Barcode Data: 201?Producing Barcode Data: 201?Barcode data anywhere, instantlyBarcode data anywhere, instantly
Data in seconds to Data in seconds to minutesminutes
Pennies per Pennies per samplesample
Link to reference Link to reference databasedatabase
A taxonomic GPSA taxonomic GPS
Usable by non-Usable by non-specialistsspecialists
• Promote barcoding as a global standard
• Build participation
• Working Groups
• BARCODE standard
• International Conferences
• Increase production of public BARCODE records
Networks, Projects, Organizations
Barcode of Life Community
Consortium for the Consortium for the Barcode of Life (CBOL)Barcode of Life (CBOL)
Established May 2004 with Sloan Foundation grantEstablished May 2004 with Sloan Foundation grantSecretariat opens at Smithsonian, September 2004Secretariat opens at Smithsonian, September 2004Now in its third two-year funding periodNow in its third two-year funding periodWorkshops, Working Groups, networking, Workshops, Working Groups, networking, representation/marketingrepresentation/marketingNow an international affiliation of 200+ members in Now an international affiliation of 200+ members in 50+ countries:50+ countries:– Natural history museums, biodiversity organizationsNatural history museums, biodiversity organizations– Users: e.g., government agenciesUsers: e.g., government agencies– Private sector biotech companies, database providersPrivate sector biotech companies, database providers
CBOL Member Organizations: 2009
• 200+ Member organizations, 50 countries
• 35+ Member organizations from 20+ developing countries
Barcode Sequence
Voucher Specimen
Species Name
Specimen Metadata
Literature(link to content or
citation)
BARCODE Records in INSDC
Indices - Catalogue of Life - GBIF/ECAT
Nomenclators - Zoo Record - IPNI - NameBank
Publication links - New species
GeoreferenceHabitat
Character setsImages
BehaviorOther genes
Trace filesOther
DatabasesPhylogenetic
Pop’n GeneticsEcological
Primers
Databases - Provisional sp.
Outreach ActivitiesOutreach ActivitiesCape Town, South Africa, April 2006, SANBICape Town, South Africa, April 2006, SANBI– Scale insects in African agricultureScale insects in African agriculture
Nairobi, Kenya, October 2006 Nairobi, Kenya, October 2006 – Commercial fisheries in Rift Valley lakesCommercial fisheries in Rift Valley lakes
Brazil, March 2007Brazil, March 2007– Hardwood tree speciesHardwood tree species– Endangered mammals, reptiles, amphibiansEndangered mammals, reptiles, amphibians
Taiwan, September 2007Taiwan, September 2007
Nigeria, October 2008Nigeria, October 2008
Beijing, May 2009Beijing, May 2009
India, March 2010India, March 2010
CBOL’s Global ProjectsCBOL’s Global ProjectsFish Barcode of Life (FISH-BOL)Fish Barcode of Life (FISH-BOL)– 30,000 marine/freshwater species by 201030,000 marine/freshwater species by 2010
All Birds Barcoding Initiative (ABBI)All Birds Barcoding Initiative (ABBI)– 10,000 species by 201010,000 species by 2010
Tephritid fruit fliesTephritid fruit flies– 2,000 pest/beneficial species and relatives by 20082,000 pest/beneficial species and relatives by 2008
MosquitoesMosquitoes– 3,300 species by 20083,300 species by 2008
Endangered speciesEndangered speciesTrees of the worldTrees of the world
Adoption by RegulatorsAdoption by Regulators
Food and Drug Administration Food and Drug Administration – Reference barcodes for commercial fishReference barcodes for commercial fish
NOAA/NMFSNOAA/NMFS– $100K for Gulf of Maine pilot project$100K for Gulf of Maine pilot project– FISH-BOL workshop with agencies, Taipei, Sept 2007FISH-BOL workshop with agencies, Taipei, Sept 2007
Federal Aviation Administration – $500K for birdsFederal Aviation Administration – $500K for birdsEnvironmental Protection AgencyEnvironmental Protection Agency– $250K pilot test, water quality bioassessment$250K pilot test, water quality bioassessment
FAO International Plant Protection CommissionFAO International Plant Protection Commission– Proposal for Diagnostic Protocols for fruit flies Proposal for Diagnostic Protocols for fruit flies
CITES, National Agencies, Conservation NGOsCITES, National Agencies, Conservation NGOs– International Steering Committee, identifying pilot projectsInternational Steering Committee, identifying pilot projects
Why barcodes are shortWhy barcodes are short
Low cost and simplicityLow cost and simplicity– Single capillary reads adequateSingle capillary reads adequate– Avoid post-sequencing IT needed for pyroseqAvoid post-sequencing IT needed for pyroseq
Adequate for taxonomyAdequate for taxonomy– Miminizes uniformative sequence lengthMiminizes uniformative sequence length– Limiting supply rate of identified specimensLimiting supply rate of identified specimens– Technology more accessible to small labsTechnology more accessible to small labs
Regulatory/inspection applications easier Regulatory/inspection applications easier
Barcoding and MetagenomicsBarcoding and Metagenomics
Lots of interest for ecology, less for regulationLots of interest for ecology, less for regulation
Pyrosequencing of environmental mixturesPyrosequencing of environmental mixtures– Construct species list from mixtureConstruct species list from mixture– Predator-prey relations from gut contents, fecesPredator-prey relations from gut contents, feces– Food-web reconstructionFood-web reconstruction
Relies on high accuracy (some species differ Relies on high accuracy (some species differ by only a few percent)by only a few percent)
Not cost-effective for single specimensNot cost-effective for single specimens
What barcode providers have What barcode providers have and are producingand are producing
770,000 records from ~100,000 species770,000 records from ~100,000 species– Agricultural pestsAgricultural pests– Water quality indicatorsWater quality indicators– Disease vectorsDisease vectors– Endangered speciesEndangered species– Commercial species of food, feed, commoditiesCommercial species of food, feed, commodities
5 million records from 500K species by 20155 million records from 500K species by 2015
International Barcode of Life Project (iBOL) International Barcode of Life Project (iBOL) Theme 1 – DNA Barcode LibraryTheme 1 – DNA Barcode Library
WG 1.1 VertebratesWG 1.2 Land PlantsWG 1.3 FungiWG 1.4 Human Pathogens and ZoonosesWG 1.5 Agricultural and Forestry Pest and Their
ParasitoidsWG 1.6 PollinatorsWG 1.7 Freshwater Bio-SurveillanceWG 1.8 Marine Bio-SurveillanceWG 1.9 Terrestrial Bio-SurveillanceWG 1.10 Polar Life
What barcode providers wantWhat barcode providers wantHigh PCR and sequencing success ratesHigh PCR and sequencing success rates
Bigger window into older, compromised Bigger window into older, compromised samplessamples
Better software integration to eliminate Better software integration to eliminate bottlenecksbottlenecks
Smaller labs/developing countries:Smaller labs/developing countries:– Lower equipment and maintenance costsLower equipment and maintenance costs– Simplification for techs with less trainingSimplification for techs with less training– Install anywhere without lab renovationsInstall anywhere without lab renovations– Willing to accept slower throughputWilling to accept slower throughput
What barcode users wantWhat barcode users wantAnswers to specific questions:Answers to specific questions:– Is this thing on this list of species or not?Is this thing on this list of species or not?– Is this thing a member of this genus/family?Is this thing a member of this genus/family?– Which of the species on this list is this thing?Which of the species on this list is this thing?– What species is this thing?What species is this thing?
Production-scale capabilities:Production-scale capabilities:– Hundreds to thousands of installationsHundreds to thousands of installations– Lower but constant throughputLower but constant throughput– Rapid turnaroundRapid turnaround– The right price-point and limited life cycle costsThe right price-point and limited life cycle costs
What barcode users would do What barcode users would do with the reference librarieswith the reference libraries
Inspection stations at every port and international Inspection stations at every port and international airport for:airport for:
– Agricultural pest controlAgricultural pest control
– Illegal trade in endangered speciesIllegal trade in endangered species
– Violations of trade quotasViolations of trade quotas
Regular Federal and State water quality surveysRegular Federal and State water quality surveys
Federal, State and local food inspectionFederal, State and local food inspection
Public health monitoring and diagnosesPublic health monitoring and diagnoses
NBII, 25 February 2009NBII, 25 February 2009
GenBank, EMBL, and DDBJGenBank, EMBL, and DDBJGlobal, Open Access to Barcode DataGlobal, Open Access to Barcode Data
http://www.insdc.org/
NBII, 25 February 2009NBII, 25 February 2009
Linkout from GenBank to BOLD
NBII, 25 February 2009NBII, 25 February 2009
Linkout from GenBank to Taxonomy
NBII, 25 February 2009NBII, 25 February 2009
Link from GenBank to Museums