PROTOCOLS FOR THE HIGHVOLUME ASSEMBLY OF DNA BARCODES

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PROTOCOLS FOR THEHIGH-VOLUME ASSEMBLYOF DNA
BARCODES

• Mehrdad Hajibabaei
• Barcode of Life Initiative
• University of Guelph
• Canada

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Barcoding Animals
Gaining Barcode Closure for Animals
10 million species x 10 barcodes each 100
million barcodes
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Barcoding Animals
Barcodes in Perspective
100 million barcodes 65 billion base pairs
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Barcoding Animals
The Time Frame
200 x 50K sequences per year
10 years to completion
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Barcoding Animals
Logistical Challenges
How do we increase production rates, lower costs
and speed analysis?
How do we recover barcodes from archival
specimens?
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The Barcoding Process
The Analytical Chain
Specimen or Tissue Sample
DNA Barcode
PCR COI
Sequence COI
Extract DNA
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The Barcoding Process
Model 1 Core Facility
Core Facility 50-100K Barcodes/Year 1M

Core Facility receives specimen samples
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The Barcoding Process
Model 2 Satellite Labs
Sequencing Facility 800K

Sequencing Facility receives PCR product
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The Barcoding Process
The Guelph Analytical Node
Sourcing Specimens
Extracting DNA
Guelph Analytical Node
DNA Amplification
Screening PCR Products
Sequencing
Barcode of Life Database
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Sourcing Specimens
Sourcing Specimens
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Sourcing Specimens
Specimen Submission Package
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DNA Extraction
DNA Extraction
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Destructive tissue sampling is the rule
DNA Extraction
Sample Material
But the amount is small
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Non-destructive methods are available
DNA Extraction
Non-Destructive Methods
Minute specimens
Type specimens
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DNA Extraction
Two Favored Methods
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DNA Amplification
DNA Amplification

• Primer Design Optimized
  Reaction

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DNA Amplification
Primer Design
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DNA Amplification
Optimized Reaction
Removes the need for PCR cleanup Lower cost by
decreasing reaction volume
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Screening PCR Products
Screening PCR Products
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Screening PCR Products
A Fast-Gel Approach
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Screening PCR Products
A Micro-Fluidic Approach
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Screening PCR Products
A Comparison of Methods
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Sequencing
Sequencing
Sequencing Reaction and Clean up
Sequence Alignment
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Sequencing
Sequencing Reaction and Clean up
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Sequencing
Sequence Alignment
Sequencher
SeqScape
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Data Management
The Need for a LIMS
NCBI
BoLD
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Cost Analysis
Cost Analysis of Barcoding
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Archived Specimens
Barcodes from Archived Specimens
Barcode fragmentation
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Full Barcodes versus Mini-Barcodes
Archived Specimens
N56
N41
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Archived Specimens
Immortalizing and Rescuing DNA
Whole genome amplification
h
DNA repair
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Archived Specimens
Whole Genome Amplification
1000-fold amplification of whole genome
Genomic DNA
Amplified Genomic DNA
Amplified Barcode Region
Whole Genome Amplification
Standard PCR
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Archived Specimens
DNA Repair
130 DNA repair genes in the human genome
DNA repair enzymes
Augmented PCR cocktails i.e. Restorase
Fragmented DNA Barcode
Repaired DNA
Amplified Barcode Region
Standard PCR
DNA repair protocol
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Archived Specimens
Early Restorase I Results
2002
1930
Range of specimen collection dates
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Archived Specimens
Early Restorase II Results
2002
1930
Range of specimen collection dates
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Partnerships
Barcodes for Biodiversity Assessment
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On the Horizon
On the Horizon
The Analytical Keychain
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On the Horizon
On the Near Horizon
Speed Specimen to barcode-based ID in 3 hours
ID Time
Volume 500K sequences per-year via automation
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Acknowledgments
Acknowledgements
Laboratory
Database
Jeremy DeWaard
Sujeevan Ratnasingham
Nataly Ivanova
Rob Dooh
Stephanie Kirk
Janet Topan
Funding Support
Angela Hollis
Gordon and Betty Moore Foundation
Collections
Dan Janzen
NSERC
John Burns
Canada Research Chairs Program
Jean-Francois Landry
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