DNA Barcoding Amy Driskell Laboratories of Analytical Biology National Museum of Natural History Smithsonian Institution, Wash. DC 1
Outline 1. Barcoding in general 2. Uses & Examples 3. Barcoding Bocas Algae 4. Data, Analysis, etc. 2
What is a DNA barcode? A fingerprint for identification of everything A short, easily and universally amplifiable, and reasonably variable piece of DNA 3
Requirements For Barcode label in GenBank, for Reference in BOLD Sequence is from a vouchered specimen Re identify Voucher meta information ti 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 4
Organizations Consortium for the Barcode of Life (CBOL) (www.barcoding.si.edu) i d Barcode of Life Database (BOLD) (www.barcodinglife.org) g) International Barcode of Life (ibol) (www.ibolprojec.org) FishBOL, All Birds Barcoding Initiative, MarBOL, etc. etc. 5
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 ti introns. 6
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 7
DNA Barcoding is an imperfect science Evolutionary History: Hybridization (plastid genomes) Pseudogenes Heteroplasmy or multiple copies (nuclear markers) Lineage sorting, recent speciation iti Slow rates of sequence divergence Practical Difficulties Lack of universality Co amplification Incomplete sampling Lack of taxonomic experts 8
Barcode of Life Database (BOLD) 9
Today s Statistics BOLD Total: 570,000 Species: 62,000 Reference : 234,000 Ref species: 13,774 GenBank Barcode keyword: 37,000 10
Algal Barcodes in BOLD 11
SI s Barcoding Philosophy Collaborate taxonomic experts, students Train students, interns, other researchers Assist labsetup setup, protocol development, collecting Archive a lending library of high h quality, wellvouchered DNAs Explore biodiversity in collaboration with taxonomists and phylogeneticists. 12
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 13
L.A.B. Examples LAB& Bocas taxonomic workshops CeDAMAR Antarctic deep sea surveys Caribbean Fish Central American Frogs Biocode Moorea 14
Practical, Fundable Uses Method of identification, particularly l useful lto non experts Import/export and pest control Fisheries management Water and habitat quality assessment Partial or fragmentary sample analysis Associating different life stages 15
L.A.B. Examples USAF/FAA Bird Strike Project Larval Fish ID US Food & Drug Agency US Enviromental Protection Agency 16
Barcoding Bocas Algae Collections 2007, 2008, 2009 ~1000 specimens Red Algae: ~250 specimens sequenced Lab protocol and primer development 17
The Process of Barcoding 1. Extraction: methods, machines, timing 18
The Process of Barcoding 2. Amplification Polymerase Chain Reaction Primers 19
The Process of Barcoding 3. Sequencing 20
The Process of Barcoding Submission and organization of metadata Quality control, data processing Accuracy checking 21
Nov. 9-13, 2009 Website: dnabarcodes2009.org 22