1. DNA Barcoding: Analytical Capacity and Approaches Lee Weigt, Smithsonian Institution, LAB Introduction SI’s LAB & barcoding efforts Portion of the workflow to be addressed Extract, amplify, sequence fast, cheap, easy clarify KNOWNS vs. UNKNOWNS who, what, where, when, why, how, how much Technology pipeline / costs / throughput

2. Smithsonian Institution National Air & Space Museum National Museum of the American Indian Several art museums & galleries, including National Portrait Gallery, American Art Museum, African Art Museum, National Museum of African American History & Culture National Museum of American History National Postal Museum SCIENCE/RESEARCH *NMNH- National Museum of Natural History *NZP - National Zoological Park *SERC - Smithsonian Environmental Research Center *STRI - Smithsonian Tropical Research Institute *SCMRE - Smithsonian Center for Materials Research & Education

3. NMNH Operations Exhibits Development Education Administration Research and Collections (ADRC) –Paleobiology –Mineral Sciences –Anthropology –Vertebrate Zoology –Invertebrate Zoology –Entomology –Botany –LAB - Laboratories of Analytical Biology Additionally: Smithsonian Marine Station in Florida and Belize

4. Strengthen our research: use new technologies to discover, organize & communicate the building blocks of the Tree of Life Mobilize collections information for diverse users 83 million specimens 250,000 type specimens Largest biodiversity collection and group of systematists in the world Components: – DNA barcoding – Digital library, imaging & web based tools – Encyclopedia of Life Enhanced Taxonomy Initiative

5. NMNH’s L.A.B. Laboratories of Analytical Biology –Genomics Core –Microscopy/Imaging Core –Isotope Core (recently “outsourced”) –Training –Data analysis/ IT –Instrumentation –Remote management of other facilities/investigators

6. Need to collect Already collected Large scale barcoding projects - where the labwork falls (keep Sujeevan happy!)

7. This talk will focus on the labwork

8. Barcoding efforts at LAB/NMNH Birdstrike IDs Birds of North America All Birds Barcoding Initiative Plant Barcoding gene Larval reef fish identification Barcoding Pipeline - automated instrumentation available for barcoding projects

9. Birdstrike DNA ID process

10. Plant barcode locus: trnH-psbA + ITS PNAS early edition 31 May 2005

11. DNA Barcoding the Inshore Reef fishes of Belize for Larval IDs Match the diffcult to identify larvae to the vouchered adults using DNA barcode sequences

12. The SIMPLE Barcoding Process Tissue Extract Amp Sequence Database Technologies are changing and improving constantly -- this talk will focus on the process WHILE we are building the “library of known sequences” - touching on doing some ID’s in the process, but not on the ultimate utilization of that library (i.e. - “fish chips”, etc) Slides/examples are NOT intended as endorsements or recommendations of specific vendors, kits, or instruments - they are merely examples of what we use.

13. Barcoding needs Genomics needs A sequencing “factory” could run the sequences on ALL-FISH (10X coverage) in less time than we will be in Guelph

14. Whous What DNA barcode all fishes Whenwe’ve already started Whereyour lab, their lab, our lab -take your pick Whybecause it’s there… Howprotocols, instruments, tracking, etc How muchper sample -- knowns vs. unknowns (how many) per protocol/method/instrument(s) CAPACITY AND APPROACHES

15. FORMAT OF PROCESS SLIDES

16. Quality, longevity - do it right Quick and dirty - do it fast and cheap Easy to repeat if needed

17. BARCODE COSTS-LAB All costs in US$ No equipment amortization No labor figured in

18. Proposed pipeline-LAB

19. Barcoding process overview

20. The LOW throughput lab environment Notice the lack of automated instruments!

21. The MEDIUM throughput lab environment Everything in 96well plate format Some automated liquid handling

22. The HIGH throughput lab environment Notice the lack of people!

23. PCR capacity=18 plates(96) simultaneously

24. DNA Extraction capacity LOW Tubes x1 MED Plates/kits x96 HIGH Automated (3x(4x96))/d Labor intensive Kits=expensive Pushbutton/ walkaway vs. flexible 4plates=4h =$0.80/sample

25. PCR & sequencing reaction setup/purification capacity LOW tubes/strips/plates HIGH automation MEDIUM Plates/384 Reaction volumes >5ul3-10ul<2ul

26. DNA sequencers 16 96 96 x #instruments Low Medium High

27. Automating high-quality DNA/RNA extractions 4 plates of 96 samples 4 hours Walk-away automation Plant or animal tissue $0.80/sample ~ $160,000 purchase

28. Automating PCR/sequencing reaction setup, cycling, purification

29. Walk-away automation PCR reaction setup thermal cycle purification Cycle Sequence reaction setup thermal cycle purification Dispense

30. Integral thermal cycler / magnet Self cleaning 96/384 well plates FAST!! Reagent savings 500nl seq rxn –Yes, that’s NANOliter, as in SUB-microliter

31. DNA sequencers 16 96 96 x #instruments Low Medium High $125K $350K $=if you have to ask…

32. DNA Barcode Project costs Old data in this slide -prices constantly coming down

33. 1 whole genome amplification + 2 biotech NDA/MTA for pre-PCR/PCR We’re going to have some fun this fall exploring the collections with the latest/greatest biotech tools to see what we can recover…

34. FISH-BOL: Take home message The LABORATORY portion of this effort is relatively: –Available/accessible –Not very time consuming (fast) –Inexpensive (cheap) –Simple and automated (easy) –Ready and waiting… Doing volume lab work? Tip: high quality primer synthesis will save $in the long run