DNA Barcoding
Amy Driskell
Laboratories of Analytical Biology
National Museum of Natural History
Smithsonian Institution, Wash. DC
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Outline
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Barcoding in general
Uses & Examples
Barcoding Bocas Algae
Data, Analysis, etc.
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What is a DNA barcode?
• A fingerprint for identification of everything
• A short, easily and universally amplifiable, and
reasonably variable piece of DNA
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Requirements
For “Barcode” label in GenBank, for “Reference” in BOLD
• Sequence is from a vouchered specimen - Re-identify
• Voucher meta-information required: GPS coordinates,
photographs (in situ, in some cases), collector and
identifier data - Re-collect
• DNA sequence is high quality (minimum length, minimum
coverage, minimum “quality scores” - Compare
• Raw data (chromatograms) publicly available - Re-analyze
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Organizations
• Consortium for the Barcode of Life (CBOL)
(www.barcoding.si.edu)
• Barcode of Life Database (BOLD)
(www.barcodinglife.org)
• International Barcode of Life (iBOL)
(www.ibolprojec.org)
• FishBOL, All Birds Barcoding Initiative, MarBOL,
etc. etc.
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Barcode “Markers”
• Animals: Mitochondrial cytochrome oxidase
subunit I (CO1/COX1) ~ 650 bp
– BUT, not variable in cnidarians, 16S rDNA historically
more informative in many groups (e.g. frogs, some
crustaceans). Second marker possible.
• Fungi: Nuclear internal transcribed spacer (ITS)
– Non-coding gene and difficult to align. But long history
in fungal studies, large existing databases, CO1
contains introns.
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Barcode “Markers”
• Red & Brown Algae: CO1
– Not nearly as “universally” amplifiable as in animals,
requires many different primers
• Green Plants: Not decided
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SI botanists promote rBCL and the trnH-psbB spacer
Others prefer for rBCL and matK
All are chloroplast genes
Green algae? Still not clear
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DNA Barcoding is an imperfect science
• Evolutionary History:
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Hybridization (plastid genomes)
Pseudogenes
Heteroplasmy or multiple copies (nuclear markers)
Lineage sorting, recent speciation
Slow rates of sequence divergence
• Practical Difficulties
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Lack of universality
Co-amplification
Incomplete sampling
Lack of taxonomic experts
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Barcode of Life Database (BOLD)
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Today’s Statistics
• BOLD
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Total: 570,000
Species: 62,000
“Reference”: 234,000
“Ref” species: 13,774
• GenBank
– “Barcode” keyword: 37,000
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Algal Barcodes in BOLD
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SI’s Barcoding Philosophy
• Collaborate - taxonomic experts, students
• Train - students, interns, other researchers
• Assist - lab setup, protocol development,
collecting
• Archive - a “lending library” of high quality, wellvouchered DNAs
Explore biodiversity in collaboration with
taxonomists and phylogeneticists.
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Biologically Interesting Uses
Biodiversity Exploration
– Discovery of sibling species
– Quick assessment of local diversity
– “DNA assisted alpha taxonomy”
• Ecological & Behavioral Studies
– Habitat assessment
– Stomach content analysis
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L.A.B. Examples
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LAB & Bocas taxonomic workshops
CeDAMAR Antarctic deep sea surveys
Caribbean Fish
Central American Frogs
Biocode Moorea
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Practical, Fundable Uses
• Method of identification, particularly useful to
non-experts
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Import/export and pest control
Fisheries management
Water and habitat quality assessment
Partial or fragmentary sample analysis
Associating different life stages
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L.A.B. Examples
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USAF/FAA “Bird Strike” Project
Larval Fish ID
US Food & Drug Agency
US Enviromental Protection Agency
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Barcoding Bocas Algae
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Collections 2007, 2008, 2009
~1000 specimens
Red Algae: ~250 specimens sequenced
Lab protocol and primer development
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The Process of Barcoding
1. Extraction: methods, machines, timing
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The Process of Barcoding
2. Amplification Polymerase Chain
Reaction
-Primers
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The Process of Barcoding
3. Sequencing
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The Process of Barcoding
• Submission and organization of metadata
• Quality control, data processing
• Accuracy checking
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Nov. 9-13, 2009
Website:
dnabarcodes2009.org
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