Academia Sinica, 16 January 2007 DNA Barcoding: An Emerging Global Standard for Species Identification Consortium for the Barcode of Life National Museum.

Academia Sinica, 16 January 2007

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


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


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


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


1. Databasing2. Labeling3. Imaging4. Tissue sampling5. DNA extraction6. PCR7. PCR check8. Sequencing reaction9. Sequencing cleanup10. Sequencing11. Trace editing & submission

Analytical chain


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


Methods


Barcode of Life Database


1. Databasing2. Labeling3. Imaging4. Tissue sampling5. DNA extraction6. PCR7. PCR check8. Sequencing reaction9. Sequencing cleanup10. Sequencing11. Trace editing & submission

Analytical chain


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


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


Methods


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



contents, droppings




Associating Life Stages, Processed Parts, Dimorphic Genders


Hypopygus lepturusHoedeman 1962


Steatogenys elegans

Steatogenys duidae

Steatogenini until the early 90’s


Nijssen & Isbrüker 1972Nijssen & Isbrüker 1972

Color patterns in Hypopygus


Hypopygus neblinaeMago-Leccia 1994




Steatogenys

Steatogenini during the 90’s






StegostenoposTriques 1997

StegostenoposTriques 1997

Steatogenys

Steatogenini during the 90’s / today


Steatogenys sp.

Hypopygus lepturus

Stegostenopos cryptogenes

R. Bernhard, 20048a8a


AA

CC

DD

Steatogenys

H. lepturusH. lepturus

RAG 1

MP/ML/DistStegostenopusStegostenopus

Hypopygus neblinaeHypopygus neblinae


12S16S Strict of

ML/MP/Dist

AA

CC

EEDD

H. neblinaeH. neblinae

StegostenopusStegostenopus

Steatogenys

H. l

eptu

rus

H. l

eptu

rus


DDEE

H.

lep

turu

sH

. le

ptu

rus

2781

2845

2876

2885

2792

2791

D-loop MP/ML/Dist


Eigenmannia sp.

COI - BARCODE MP

H. l

eptu

rus

H. l

eptu

rus

AA2781

2845

CC 2792

2791

DD

EE

H. neblinaeH. neblinae

StegostenopusStegostenopus



contents, droppings




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


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


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 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


Infrastructure of Taxonomy:Fragmented, 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


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


Barcode Sequence

Voucher Specimen

Species Name

Specimen Metadata

Literature(link to content or

citation)

BARCODE records in GenBank

Indices - Catalog 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


Digitizing Taxonomic Literature

• CBOL’s 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








The Barcode Assembly Line: 2006

Freshly collected specimens

Frozen tissue Young museum specimens

DNA Barcode Data


The Barcode Assembly Line: 2008Opening the museum treasure-trove

Freshly collected specimens

Frozen tissue Young museum specimens

DNA Barcode Data

Formalin-fixed specimens

Older museum specimens


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








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)






• Plants: Identify barcode gene region(s) for land plants


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


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


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


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


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


Milestones for 20082007 20082006

Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2

International Conference

Development of Consensus Plant Barcode Region

Data Analysis Protocols and S/W

Formalin Study

Advanced Lab Protocols

200K records 500K records100K records

Demonstrator System Launched

Database:

Data Analysis WG:

DNA WG:

Plant WG:

Database WG: Extended DB Interoperability

BoLI Data Portal Launched

Campaigns: Regional Groups Operational

First Data Releases

10K birds30K fish

Data Standards

Academia Sinica, 16 January 2007 DNA Barcoding: An Emerging Global Standard for Species Identification Consortium for the Barcode of Life National Museum.

Documents

academia sinica

genbank slide

barcode of life database

sequencing cleanup

dna barcodes

dna extraction

producing barcode data

pcr check