high throughput plasmid sequencing with illumina and clc bio · plant transformaon seed ... clc bio...
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High Throughput Plasmid Sequencing with Illumina and CLC bio
Ajay Athavale Monsanto Company 05 June 2012
6/4/2012 1 Monsanto Company Confidential
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Monsanto’s Transgenic Plant Pipeline: Sequencing 1,000’s of plasmids per year
Hypothesis
Nomina/on
Cloning
Sequencing Plant Transforma/on
Seed Genera/on
Efficacy Tes/ng
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Increasing Clone Complexity Required New Solutions for Plasmid Sequencing & Analysis
• NextGen sequencing offers significant savings & reduced processing time • Illumina was demonstrated to be optimal NGS platform for plasmid sequencing • Scalable and accurate finishing tools were needed for Illumina sequence reads
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0%
20%
40%
60%
80%
100%
120%
2007 2008 2009 2010 2011 2012‐Sgr 2012‐Ilmn
% of C
lone
s
Clone Complexity & Sequencing Costs
Simple Moderate Complex Total Cost
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Requirements for HTP Plasmid Finishing Platform Performance, Accuracy & Scalability
Support structurally challenging plasmids Support for multiple sequencing platforms Hybrid assemblies (across platforms) User configurable & flexible parameters
• Read trimming • Mutation detection (SNPs) • Insertion & Deletion detection (DIPs)
Usability & User Interface Easily edit/manipulate assemblies Execution from both UNIX and GUI environments User friendly visualization tools
Integration with in house corporate research data systems
Customization of finishing workflows
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CLC bio was chosen as next gen finishing tool, given these criteria
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Demonstrated identical finishing results for structurally complex plasmids using Illumina and CLC Bio
Plasmid Known Complex Sequences
Observed Seq Sanger + CONSED
Observed Seq Illumina + CLC bio
Plasmid A None
Plasmid B Mixed PopulaDon
Plasmid C Homopolymer tracts
Plasmid D Homopolymer tracts Tandem Repeats
Plasmid E Homopolymer tracts Inverted Repeats
Plasmid F Homopolymer tracts Inverted Repeats Tandem Repeats
Plasmid G Large InserDon
Plasmid H Large DeleDon
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MiSeq Sequencing
Assembly &
Analysis
Overview of the Current Sequencing & Finishing Process with Illumina and CLC bio
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Glycerol Receipt
Plasmid DNA IsolaDon
Clone Re‐array & DNA NormalizaDon
Nextera Library Prep Library QuanDficaDon & Pooling
Assembly & Analysis in CLC bio
Review Sequence
Advance Perfect Clones
Colony PropagaDon
DNA Prep
Library Prep &
Sequencing
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Automated Assembly & Analysis Workflow in CLC bio streamlines workflow • The CLC bio Genomics Server and Command line tools
enable efficient parallel processing of large batches of constructs
Read Mapping
Coverage Analysis
SNP Detection
DIP Detection
Read Mappin
g
Prepare Config file
Import Illumina data
Quality Trimming
Read Mapping
Coverage Analysis
SNP DetecDon
DIP DetecDon
Review Assemblies
• For the non‐UNIX user, command line tools can be easily run in the GUI (CLC Genomics workbench)
Assembly & Analysis Workflow
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Identification of low coverage regions with the “Contig Analysis” tool
Easy identification of low coverage areas
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The “Primer Creator” tool helps to streamline efforts to close gaps
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Design primers around
annotated regions
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Simplified variant detection with the “SNP Detection” tool
Workflow specific op/miza/on of SNP calling parameters is cri/cal for accuracy!
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“DIP Detection” for Deletions & Insertions
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Manual review is s/ll required as the
algorithm does not always detect large
inser/ons and dele/ons, leading to a
mis‐assembly...
The de novo assembly tool helps to resolve large inser/ons or dele/ons!
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Denovo assembly used to resolve Indels
Challenging plasmids are typically able to be resolved using the de novo assembly workflow
de novo assembly is easier than tearing & joining conDgs manually, typically equally as effecDve
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CLC Bio Meets Requirements for Monsanto HTP Plasmid Finishing
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Performance, Accuracy & Scalability Support structurally challenging plasmids Support for multiple sequencing platforms Hybrid assemblies (across platforms) User configurable & flexible parameters
Read trimming Mutation detection (SNPs) Insertion & Deletion detection (DIPs)
Usability & User Interface
Easily edit/manipulate assemblies Execution from both UNIX and GUI environments User friendly visualization tools
Integration with in house corporate research data systems Customization of finishing workflows
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Additional CLC bio capabilities are also used across Monsanto
• In silico cloning & plasmid design
• Sanger sequence review/assembly
• Plasmid sequencing & finishing via Sanger & Illumina
• Genome assembly for microbes and plants
• DNA and Protein alignment for research teams
• … and much more!
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Acknowledgements
• CLC Bio – Jannick Bendtsen – Henrik Sandmann – Joe Salvatore
• Monsanto – Todd Michael – Dan Ader – Amber Ford – Susan Johnson – Cynthia LaBanca – Kim Lawry – Jing Lu – Karen Martin – Tim Mitsky – Stacie Norton
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