Methods of High Volume Barcode Assembly

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Methods of High Volume Barcode Assembly
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Barcode Analytical Challenges
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GATHERING BARCODE RECORDS
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Analytical Chain
Specimen
Collection Data
Web-Accessible Data andDNA Barcode
Tissue Sample
Photograph
ExtractDNA
PCR Amplify
Sequence
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Specimen Imaging CCDB Advances
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Specimen Handling and Shipment
Specimen Submission Package
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Data Entry
Barcode Analysis Lab Book
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Analytical Chain
Specimen
Collection Data
Web-Accessible Data andDNA Barcode
Tissue Sample
Photograph
ExtractDNA
PCR Amplify
Sequence
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Barcode Analytical Steps
Lysis
Cycle Sequencing
Extraction
Sequence
PCR Screen
PCR
PCR
Trace Sub
Sample Submission
Analysis
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Barcode Production Canadian Centre for DNA
Barcoding
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Production
Production for February 2007
First Pass Success 91
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Facility Canadian Centre for DNA Barcoding
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CCDB STAFF

• 4 Investigators
• 2 Molecular Scientists
• 6 Postdoctoral Fellows
• 7 Core Lab Staff
• 7 BOLD Team
• 4 Collection/Digital Media

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Tissue Sampling and Lysis
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Analytical chain

• DNA extraction
• placing tissue sample in lysis solution
• two strategies
• Cheap, non-archival,
• Expensive, higher quality

Method Cost () Sensitivity
Chelex 0.34
Membrane 2.00
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DNA Extraction
DNA Extraction
50 cents per sample
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DNA Extraction
DNA Extraction
Centrifuge/Manual Method
Robotic Method
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DNA Amplification
DNA Amplification
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Screening PCR Products
A Fast-Gel Approach E-Gel 96
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Platinum Taq Enhancing PCR Success
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Optimizing PCR
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Cycle Sequencing Reaction
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Analytical chain

• Sequencing reaction
• no PCR cleanup
• smaller volumes, BigDye reduction
• premixed, frozen

Sequencing cleanup
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Sequence Clean up
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Sequence Clean up
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Sequence Read
Standard Bidirectional Reads
3X ABI 3730XL
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Sequencing Capacity

• ABI 3730XL Sequencer
• 500,000 sequences per year
• 250,000 specimens per year

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Trace Processing

• Trace editing submission
• SeqScape Sequencher
• submission to BOLD
• automated system a component of BOLD-LIMS

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Archiving DNA Extracts

• Whatman FTA cards
• archiving, transporting
• plain filter paper

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Barcode Analytical Elements
Lysis
Cycle Sequencing
Sequence
Extraction
PCR Check
PCR
PCR
Trace Sub
Sample Submission
Analysis
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Linking the Analytical Chain Laboratory
Information Management System (LIMS)
PCR Check
Lysis
Cycle Sequencing
Sequence
Extraction
PCR 1
PCR 2
Trace Sub
Sample Submission
Analysis
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CURRENT OVERVIEW
Costs and Time

  Fresh/Frozen Time
Tissue Sampling  0.41  10
DNA Extraction 0.34 10
PCR Amplification 0.24 20
PCR Product Check 0.35 5
Cycle Sequencing 1.04 30
Sequencing Cleanup 0.32 5
Sequence 0.40 35
Total 3.10 115 min
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Canadian Centre for DNA Barcoding - A Venue For
Collaboration
CaseBirds of Argentina The Challenge Assemble
DNA barcodes for 1000 birds. The Result After
two weeks, all specimens were barcoded and
databased on BOLD.
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Next Generation Barcode Analysis
A Field Guide for the Third Millennium
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May 1954 Popular Mechanics
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Next Generation Barcode Analysis
Microfabricated bioprocessor for integrated
nanoliter-scale Sanger DNA sequencing
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Next Generation Barcode Analysis
Environmental Barcoding
Massively Parallel Pyrosequencing One reaction
250,000 sequence reads of 200 bp
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Next Generation Barcode Analysis
Massively Parallel Pyrosequencing
1.6 million wells (70 x 75 mm2)

• One DNA bead and hundreds of enzyme beads per
  well
• Sequencing by synthesis
• Product detection by photon emission

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DNA Barcodes By 2020
Gaining Barcode Closure for Animals
Gaining Closure for All Animals
10 million species x 10 barcodes each 100
million barcodes lt500M
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Barcode of Life International Collaborative
Initiative (2009-2014)
Goal 5M Barcode Records 500K Species 100M (2009-
2014)
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Critical Components - Reaching the 0.5M milestone