Get documents about "Methods for Streamlining High Throughput DNA Barcoding
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Document Sample
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
