High Throughput Plasmid Sequencing with Illumina and CLC bio

Ajay Athavale Monsanto Company 05 June 2012

6/4/2012 1 Monsanto Company Confidential

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 

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 

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 

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! 

“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! 

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

  

  

 

 

 

 

 

  

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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