DNA Barcoding

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DNA Barcoding

• Amy Driskell
• Laboratories of Analytical Biology
• National Museum of Natural History
• Smithsonian Institution, Wash. DC

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Outline

• 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
• 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
• Hybridization (plastid genomes)
• Pseudogenes
• Heteroplasmy or multiple copies (nuclear markers)
• Lineage sorting, recent speciation
• Slow rates of sequence divergence
• Practical Difficulties
• Lack of universality
• Co-amplification
• Incomplete sampling
• Lack of taxonomic experts

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Barcode of Life Database (BOLD)
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Todays Statistics

• BOLD
• 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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SIs Barcoding Philosophy

• Collaborate - taxonomic experts, students
• Train - students, interns, other researchers
• Assist - lab setup, protocol development,
  collecting
• Archive - a lending library of high quality,
  well-vouchered 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

• 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
• 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

• USAF/FAA Bird Strike Project
• Larval Fish ID
• US Food Drug Agency
• US Enviromental Protection Agency

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Barcoding Bocas Algae

• Collections 2007, 2008, 2009
• 1000 specimens
• Red Algae 250 specimens sequenced
• Lab protocol and primer development

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The Process of Barcoding

• 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