DNA Barcoding: Analytical Capacity and Approaches Lee Weigt, Smithsonian Institution, LAB

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DNA Barcoding Analytical Capacity and
ApproachesLee Weigt, Smithsonian Institution, LAB

• Introduction
• SIs 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

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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

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NMNH

• 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

• Operations
• Exhibits
• Development
• Education
• Administration

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Enhanced Taxonomy Initiative

• 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

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NMNHs 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/investigator
  s

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Large scale barcoding projects - where the
labwork falls
Already collected
Need to collect
(keep Sujeevan happy!)
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This talk will focus on the labwork
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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

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Birdstrike DNA ID process
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Plant barcode locus trnH-psbA ITS
PNAS early edition 31 May 2005
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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
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The SIMPLE Barcoding Process
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 IDs in the
process, but not on the ultimate utilization of
that library (i.e. - fish chips, etc)

• Tissue
• Extract
• Amp
• Sequence
• Database

Slides/examples are NOT intended as endorsements
or recommendations of specific vendors, kits, or
instruments - they are merely examples of what we
use.
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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
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Who us What DNA barcode all fishes When weve
already started Where your lab, their lab, our
lab -take your pick Why because its
there How protocols, instruments, tracking,
etc How much per sample -- knowns vs.
unknowns (how many) per protocol/method/instru
ment(s) CAPACITY AND APPROACHES
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FORMAT OF PROCESS SLIDES
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Quality, longevity - do it right
Quick and dirty - do it fast and cheap
Easy to repeat if needed
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BARCODE COSTS-LAB
All costs in US No equipment amortization
No labor figured in
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Proposed pipeline-LAB
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Barcoding process overview
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The LOW throughput lab environment
Notice the lack of automated instruments!
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The MEDIUM throughput lab environment
Everything in 96well plate format Some
automated liquid handling
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The HIGH throughput lab environment
Notice the lack of people!
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PCR capacity18 plates(96) simultaneously
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DNA Extraction capacity
LOW Tubes x1
MED Plates/kits x96
HIGH Automated (3x(4x96))/d
4plates4h 0.80/sample
Pushbutton/ walkaway vs. flexible
Labor intensive
Kitsexpensive
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PCR sequencing reaction setup/purification
capacity
LOW tubes/strips/plates
HIGH automation
MEDIUM Plates/384
Reaction volumes gt5ul 3-10ul lt2ul
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DNA sequencers
Low
Medium
High
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96 x instruments
96
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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
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Automating PCR/sequencing reaction setup,
cycling, purification
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Walk-away automation

• PCR
• reaction setup
• thermal cycle
• purification
• Cycle Sequence
• reaction setup
• thermal cycle
• purification
• Dispense

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Integral thermal cycler / magnet

• Self cleaning
• 96/384 well plates
• FAST!!
• Reagent savings
• 500nl seq rxn
• Yes, thats
• NANOliter, as in
• SUB-microliter

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DNA sequencers
Low
Medium
if you have to ask
High
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96 x instruments
125K
96
350K
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DNA Barcode Project costs
Old data in this slide -prices constantly coming
down
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1 whole genome amplification 2 biotech NDA/MTA
for pre-PCR/PCR
Were going to have some fun this fall exploring
the collections with the latest/greatest biotech
tools to see what we can recover
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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