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DNA BarcodingDNA BarcodingAmy Driskell

Laboratories of Analytical BiologyNational Museum of Natural HistorySmithsonian Institution, Wash. DC

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Outline

1. Barcoding in general2. Uses & Examples3. Barcoding Bocas Algae4. 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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Today’s 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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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, 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

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, 2009Website: dnabarcodes2009.org