Utah State University – 29 Nov 2006 DNA Barcoding: An Emerging Global Standard for Species...

Utah State University – 29 Nov 2006

DNA Barcoding: An Emerging Global Standard for Species

IdentificationConsortium for the Barcode of Life

National Museum of Natural HistorySmithsonian Institution

http://www.barcoding.si.edu202/633-0808; fax 202/633-2938

http://www.barcoding.si.edu/


A DNA barcode is a short gene sequence

taken from standardized portions

of the genome, used to identify species

The Mitochondrial Genome

Cyt bCyt b

D-Loop

ND5

H-strand

ND4

ND4LND3

COIII

COICOIL-strand

ND6

COI

ND2

ND1

COII

Small ribosomal RNA

Large ribosomal RNA

ATPase subunit 8

ATPase subunit 6


Uses of DNA BarcodesApplied 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


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


Reactions to Barcoding: 2004• From ecologists and other users:

“This is what we need! How soon can we get started?”

• From traditional taxonomists:“Species should be based on lots of characters, not just barcodes”

• From forward-looking taxonomists:“Using molecular data as species diagnostics isn’t

new, but standardization and broad implementation are great!”

• From barcoding practitioners:“I had my doubts at the beginning, but it really works

as a tool for identification (96% accurate in a recent mollusc paper) and it is at least as good as traditional approaches to discovering new species.”


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


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• BUT…Barcoding can help to create a

21st century research environment for taxonomy

Barcode Sequence

Voucher Specimen

Species Name

Specimen Metadata

Literature(link to content or

citation)

BARCODE Data Standard

Indices - Catalog of Life - GBIF/ECAT

Nomenclators - Zoo Record - IPNI

NameBank

Publication links - New species

GeoreferenceHabitatCharacter setsImagesBehaviorOther genes

Trace files

Other Databases

PhylogeneticPop’n GeneticsEcological

Primers

Current Norm: High throughput

ABI 3100 capillary

automated sequencer

Large capacity PCR and

sequencing reactions


Fresh/Frozen Museum

Tissue Sampling $0.41 $0.41

DNA Extraction $0.34 $2.00

PCR Amplification $0.24 $0.48

PCR Product Check $0.35 $0.70

Cycle Sequencing $1.04 $2.08

Sequencing Cleanup $0.32 $0.64

Sequence $0.40 $0.80

Total: $3.10 $7.11

Cost of Reagents and Disposables

Producing Barcode Data: 2008 Faster, more portable: Hundreds of samples per hour

Integrated DNA microchips Table-top microfluidic systems


Reference vs. Micro-Barcodes

• BARCODE reference records– Adhere to data standards– Bidirectional reads, 500+ bp long– Linked to voucher, species name

• Query barcode records– Used in BLAST or other searches– Often single pass reads– Often very short – 100+ bp for good IDs– Can cost less than $2, take less than 6 hours


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


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 Org’s, 40 countries, 6 continents– Natural history museums, biodiversity organizations– Users: e.g., government agencies– Private sector biotech companies, database providers

CBOL Member Organizations June 2006: 120 Member Organizations, 40 countries


CBOL’s Underlying Principles• Standardization, cost-effectiveness

• Minimalism (scientific) – short sequence

• Global participation

• Tangible, realistic goals, near-term results

• Respond to applied user communities

• Minimalisim (organizational) – Coordination/Facilitation of Bottom-Up activities, build on existing activities, avoid new ones


CBOL’s Working Groups

• Database: Designing/constructing the Barcode Section of GenBank

• DNA: Protocols for formalin-fixed and old museum specimens; advice to new labs

• Data Analysis: Beyond phenetic methods; population genetics perspective

• Plants: Identify gene region(s) for barcoding


Outreach to Developing Countries

• 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 Asia, 2nd quarter 2007

• Second International Barcode Conference– Singapore, 13-15 June 2007

• Support from CBOL, host governments and international development agencies


Goals of Regional Meetings

• Raise awareness

• Explore potential applications in the region

• Assess greatest needs and opportunities in the region

• Identify highest priorities, construct national and regional action plans

• Start intra-regional networks and intercontinental partnerships


Current and Planned Projects

• Four Working Groups

• FishBOL and All Birds Initiatives

• Developing “Demonstrator Systems by 2008

• African Scale Insect Barcoding Initiative (planned at Cape Town Regional Meeting)

• International Network for Barcoding Invasive and Pest Species (INBIPS)

• Forming a Conservation Committee


Demonstrator DevelopmentGOAL: By June 2008, create an operational

system for global ID of an important group• Mosquitoes (disease vectors)

– Barcode 85% (2932) of 3449 known species– Minimum 5 specimens per species– Reliance on archival collections

• Tephritid fruit flies (agricultural pests)– Barcode 2000 species of 4500, 5 per species– All pests and beneficials, with closest relatives– Invasive non-destructive sampling of existing

collections


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


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

IDS – Identification System

Linkage to Mapping System


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

DNA from identified voucher

Create BARCODE reference record

ID unknownsRefine taxonomy of group

DNA from identified adult

voucher

Create BARCODE reference records

Associate immatures with names

ID unknowns

Refine taxonomy of group

DNA from unidentified immature specimen

Repository of provisional vouchers

Add names to vouchered immatures

Utah State University – 29 Nov 2006 DNA Barcoding: An Emerging Global Standard for Species...

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