Methods for Streamlining High Throughput DNA Barcoding by gjjur4356

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									   Methods for Streamlining
High Throughput DNA Barcoding
Natalia V. Ivanova, Chris M. Grainger, Alex V. Borisenko
Automation at BIO

                                       New equipment:
                         •   2 Biomek FXP robots – dual-bridge systems
                             (96-tip head and Span-8), vacuum manifold,
                             orbital shaker, tip wash station, refillable
                             reservoirs, Cytomat hotel, barcode reader

                         •   3730xl DNA sequencer




           Upgrades:
 •   Biomek NX – plate/tips stacker
     and new version of software

 •   Upgrade of existing 3730 to
     3730xl (to 96 capillaries)
Adoption of existing protocols in a new environment

                                    • Single source reagent stocks
                                    • Lab policies

        ?      Lep
              100 1
            21/1 µM
                   R

                0/20
                     06
                                    • Optimized workflow




 • Standard Operating Procedures
 • Increased QA/QC measures

 • Batch oriented production
 • Audit tracking
Outline of the analytical chain
                                                              CORE LAB PROCESSES
                                   PRE-LAB                                                                  POST-LAB
                                 PROCESSING                                                                PROCESSING
                                                                                   BOLD Specimen
                                                                                   Data Download
   Tissue Array Map,
    e.g., Box Record                   Data conversion                        Lab Book with                    Electronic
                                                                               Plate Record Archival           Lab Books
      Arrayed Tissue              Arrayed                      Lysis Plate with                                 Archive
                                                Subsampling
      Samples, e.g.,               Tissue                        94 Samples
        Matrix box                Samples
                                                                  Tissue Lysis

                                                                 DNA Extraction
                                     Tissue
                                                                   (Glass Fiber)
                                    Sampling
                                                                                                              Archive of
   Permanent                      Processed                      DNA Extracts             Archival            Residual
   Collection          Loaning                                                                                  DNA
                                  Specimens
   Repository                                                   PCR Amplification                              Extracts

                 BOLD Image
                                   Specimen                      PCR Products         Decision
                 Submission
                                    Imaging                                             step
                                                                 PCR Gel Check
                                                                                                            Analysis of
                                  Specimen                     Cycle Sequencing
                 BOLD Data                                                                                   Results
                 Submission        Prep and
                                  Databasing                   Sequenced Plates                        BOLD
   Specimens                                                                                          Sequence
                                  Unprocessed                  Sequencing Clean-up
   with Field     Accessioning                                                                       Submission
     Labels                        Specimens                                                                  Aligned
                                                                  Trace Files          Editing/Alignment     Sequence

                                                                BOLD Trace File                            BOLD Sequence
                                                                 Submission                                 Submission
Molecular protocols outline

                                 CORE LAB PROCESSES

                                                    BOLD Specimen
                                                    Data Download

              Data conversion                      Lab Book with
                                                    Plate Record
                                Lysis Plate with
                                  94 Samples

                                   Tissue Lysis

                                   DNA Extraction
                                                                             LIMS
                                     (robotic or
                                       manual)

                                  DNA Extracts             Archival

                                 PCR Amplification

                                                                      Decision
                                  PCR Products
                                                                        step
                                  PCR Gel Check

                                Cycle Sequencing

                                Sequenced Plates
                                Sequencing Clean-up


                                   Trace Files          Editing/Alignment


                                 BOLD Trace File           Trace auto-assembler
                                  Submission
Policies – processing pipelines


                                                            ‘Shot-gun’ approach for sequencing
                                                                     No spec analysis
                                                                     No PCR clean-up

                                                            Material Type
                     Variable Low Number                                Established Pipeline               Variable High Number
                    And “Odd-Ball” Material                                High Success                           Material

   Good Success (threshold       Poor Success (threshold value,   Process Plates and Failure Track
                                                                                                           Evaluation of Success
 value to “push plate through”     alternate method employed         At End of Project (hit-pick)

      Failure Track With               Process Plate with
                                                                                               Positive Hit-Pick         Negative Hit-Pick
    Other “odd ball” Plates            Alternate Method


                                                                                                     Sequence           Process Plate with
                                                                                                     Positives           Alternate Method



         Threshold Value for “Pushing Plate” is 73 Bands


          Processing can quickly become extremely costly and time
            consuming without policies and infrastructure to support
                pipelines (Robotics/Lab book system or LIMS)
Electronic Lab Book




                              Tracking of analytical history
                                  of one 96-well plate
                      •   Built-in data conversion tools, graphical interface
                      •   Retrieval of BOLD Process ID
                      •   Multiple PCR and multigene support
                      •   Evaluation and scoring of E-gel® PCR results
                      •   Output for DNA sequencer and robotic hit-picking
                      •   Plate assembly from different sources
                      •   Trace submission module
                      •   FASTA-MEGA conversion module
DNA extraction




                             Milestones
     •   Extraction protocol for vertebrates (manual & Biomek NX)
     •   Extraction protocol for insects
     •   Automation and optimization on Biomek FXP




          Cost – 50 c/sample
  Replaced Chelex and commercial kits
PCR amplification




                                       •   Trehalose acts as PCR
                                           enhancer and cryoprotector
                                       •   Robotic dispense
                                       •   Each batch is recorded in the
                                           system and labelled


       LepF1/LepR1, 01/05/07
                      Mamm, 30/04/07
                                            Fish, 04/05/07
PCR amplification




                    100
    PCR success %




                     80                              Taq experiment 1
                     60                              Taq experiment 2
                     40                              Platinum Taq experiment 1
                     20                              Platinum Taq experiment 2
                      0
                          Plate 1         Plate 2




                                    •   Platinum Taq is stable at room temperature
                                    •   Requires less optimization
                                    •   Yields much higher success rates
                                    •   Withstands freezing in mixes with trehalose
PCR amplification




                    M13-tailed primers and degenerate
                              primer cocktails
              •     Broad taxa coverage
              •     Better overlap for bi-directional reads
              •     Facilitate high throughput sequencing
              •     Savings on PCR reagents and labor
PCR amplification




                    Bidirectional contig assembly
             Regular primers




             M13-tailed degenerate cocktail
Sequencing cleanup

                                  Transition from semi-automated
                                    Sephadex to fully automated
                                        Agencourt CleanSEQ




       • 5 min per plate
        • 1/24 BigDye
      • Reduced labour
      • 1 box of tips/day


          SPRI technology


                        Image from
                      Seradyn web-site
Robotics: To Automate or Not To Automate

                     Pros                                          Cons
   Reduction of labor costs and or re-allocating Large capital/Start-up cost
   resources to other areas
   Decrease in errors during processing (e.g.    Learning curve for staff and requirements of
   ability to hit-pick) and pre-fabrication of   maintaining technical support for
   consumables (e.g. Plate Making)               instrumentation
   Ability to automate nearly every aspect of    Choose carefully, industry support for
   lab processes (many commercial products       platform is critical!
   designed for robotic platform)
   Increased production levels                   Operational capital to support robotics (i.e.
                                                 service contracts)


     Options for robotic method development:

     •   Purchasing methods with kits
     •   Asking for technical support and customized solutions
     •   Training opportunities, user forums
     •   Writing ‘in house’ methods (requires dedicated staff)
Automation vs. manual production


                                All procedures described here
                              could be done manually to ensure
                                      built-in redundancy:
                         •   Matrix boxes/plates
                         •   Deep bucket and multi bucket centrifuges
                         •   E-gels
                         •   Electronic multichannel pipettors
                         •   Number of thermocyclers
                         •   High throughput sequencers (3730xl)
                         •   Electronic lab book/LIMS to manage data flow
Cost effective production


                             •   Designing the workflow
      Methods     Workflow
         &           &       •   Defining analytical pipelines
      Materials    Policy
                             •   Setting up lab policies and SOPs

          Infrastructure     •   Minimizing reaction volumes and
                                 volumes of core reagents (units of
                                 Platinum Taq, BigDye dilution)

  Savings on labor           •   Developing ‘in-house’ approaches
                                 (e.g. DNA extraction)
  cost as a result of
 optimized workflow          •   Batch oriented production – frozen
                                 PCR and sequencing plates
   and integrated
     automation              •   Human resources – staff training
Outcome
                            Change in Amount of Labour Time
                             Requiered for Routine Lab Work

                 200

                 150
    Time (Min)




                 100

                 50

                  0                                                                                           Re-allocation of resources
                        Pre-CCDB Methods           Current CCDB Methods
                                                                                                                    with automation
                                                                                                                       Imaging and databasing
                                                   Section A: Reception
                                                   of Specimens                                                   Tissue sampling and sub-sampling
 Influence of Infrastructure Costs
 On Overall DNA Barcoding Costs                    Section B: Tissue
                                                   Lysis
                                                                                                                        Reagents preparation
                                                   Section C: DNA
                                                                                                                     Data editing and submission
                                                   Extraction

                       6%     7%                   Section D: PCR
                                   3%              Amplification/DNA
                                                   Transfer
                                         11%
                                                                                                                      Change in Specimen Production Capacity
                                                   Section E: PCR Check
                                                                                                                            with Streamlined Methods
                                                                           Production (Specimens/Year)



                                           4%      Section F: Cycle                                      250000
                                              3%   Sequencing/PCR
                                                   Product Transfer                                      200000
                                                   Section G: Sequencing
                                          8%       Clean-Up                                              150000
  51%

                                         5%        SectionH: Sequencing                                  100000
                                    2%
                                                                                                          50000
                                                   Section I: Technician
                                                   and Service Contracts                                     0
                                                                                                                     Pre-CCDB Methods        Current CCDB Methods
                                                   Section J:
                                                   Miscellaneous Costs
Acknowledgements




             Agata Pawlowski
              Becky Cowling
              Shana Hayter
                Katy Hind
               Kate Crosby
               LiuQiong Lu

                                Rick Turner
                               Becky Cowling

								
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