the history of dna sequencing technology. arc biotechnology platform dr jasper rees...
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The History of DNA Sequencing Technology
ARC Biotechnology Platform
In 2010, the ARC set out to create a new single integrated platform for advanced high
throughput, next generation technologies
Dr Jasper Rees
Vision
To create a world class biotechnology platform to lead research in agriculture in Africa
Mission• To provide excellent biotechnology research and service
platforms to enhance food security and promote agriculture in Africa
• To provide biotechnology tools in support of all areas of research and development within the mission of the ARC
• To create a world class research and innovation environment to attract the best researchers and students
• To train the next generation of specialist researchers in cutting edge biotechnologies
The Objectives
• To create Service, Development and Research structures in support of and in collaboration with all ARC Institutes and external partners
• To address the needs of commercial and emerging agriculture nationally and continentally
• To train new researchers and postgraduates at the highest level
• To compete at the top level internationally
• To have the “best in class” resources for modern biotechnology
• To raise funds external funding, nationally and internationally
Service and Research Facilities Model
Core Services
Development group
Research teams within
platform
Unit
Research teams at Institutes
Technology Focus of Unit
Key technologies being performed with the sub-platform as services to research groups and pipelines with the ARC and to outside clients
Each sub-platform to have a development group responsible for introducing new technology and applications into the core services
Internal Research teams, lead by SR, SpR, PR level scientists. With MSc, PhD and PD level students as the major part of each team. Primarily externally funded with competitive grants
External Research teams, led by SR, SpR, PR level scientists. MSc, PhD and PD level students are the major part of each team. Primarily externally funded with competitive grants
Biotech Platform Phase I – 2010/11
DNA Isolation
Next Gen Sequencing
Genotyping
Genetic Mapping
Development group
Research teams within
platform
Genomics
Sample Preparation
2D PAGE
MALDI-MS
LC/GC-MS-MS
Development group
Research teams within
platform
Proteomics
Databases
Annotation
Assembly
Expression Analysis
Development group
Research teams within
platform
Bioinformatics
Tissue Culture
Construct development
Transformation
Regeneration
Development group
Research teams within
platform
Plant Transformation
Biotech Platform Phase II/III: 2012-2015
Reverse Genetics
Gene identification
Vector Development
Construct development
Functional Genomics
Robotic platform
High throughput DNA Isolation
Genotyping
Informatics
Mutation Breeding
Marker Assisted Breeding
Robotic platform
High throughput imaging and
sensing
GC MS/MS
Informatics
Plant Phenotyping
Gene Silencing
Zinc Finger Nucleases
Gene targeting
Male Sterile Technology
Targeted Mutagenesis
Genome Engineering
Platforms and ProgramsDrought Tolerant Sorghum
Apple Breeding
Vaccine Development
Animal Reproductive
Biotechnologies
Diagnostics
Genomics 1 1 1 2 1
Proteomics 1 2 2 2 1
Bioinformatics 1 1 1 2 1
Plant Transformation 2 3
Functional Genomics 2 3 3
Genome Engineering 3 4 3
Imaging 1 2
NanoBiotechnology 2
Protein Expression 2 2
ARC Biotech Platform Implementation 2010/11
• Staff recruitment – 6 researchers team leaders in genomics, proteomics, bioinformatics; 3 more posts in 2011, more in the future.
• >25 postgraduate and postdoctoral researchers already
• Capital equipment procurement: >R10m to date• Sites in Pretoria and Stellenbosch• Building renovations in progress • Operational start: Jan 2011
ARC Biotech Platform Implementation 2010/11
• Staff recruitment – 6 researchers team leaders in genomics, proteomics, bioinformatics; 3 more posts in 2011• Jasper Rees: Genomics and Bioinformatics: Fruit• Bongani Ndimba: Proteomics: Cereals and Fruit• Damaris Odeny; Genetics, Genomics: Vegetables• Joseph Mafofo: Genomics, plant pathogens: Fruit• Dirk Swanevelder: Genomics: Plant Pests, Cereals• Farai Muchadeyi: Genotyping: Animal Genetics
Genomics Applications
Sequencing applications
• Genomes, • De novo and re-sequencing• SNPs and CNV calling
• Transcriptomes, • De novo and re-sequencing• SNPs and splicing variation• Expression profiling
• Small RNA• discovery and expression
analysis
SNP applications
• GWAS • Association Genetics• SNP validation• Candidate Genes• Diversity studies
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Platform Synergy for Agrigenomics Research
HiSeq 2000 iScan/HiScan SQ BeadXpress
Interrogate Validate/ScreenDiscover
► De novo Genome Sequencing
► mRNA Sequencing & Gene Expression
► SNP Discovery
► DNA Methylation, Targeted Resequencing
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► Genome-wide marker / phenotype association
► Focused marker / phenotype association
► Genetic Identity Confirmation
► QTL Mapping Disease Screening►
Marker Assisted Selection►
Parentage / Lineage►
Genetic Purity Screening►
110102103104105106107108>109
Discovery Focused Research/Validation Screening
Whole Genome Single Marker
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Illumina HiScanSQ Platform
• Dual function system• SNPs and Sequencing
• High throughput SNPs• Infinium and GoldenGate technology• 10k to 2.5M SNP chips
• Next Generation Sequencing• Linear scanning optics• 100 Gb of DNA sequence per run• Paired end sequencing• 400 M filtered clusters per flow cell• 8 lanes per flow cell• Sample Multiplexing
DNA(0.1-1.0 ug)
Single molecule arraySample
preparation Cluster growth5’
5’3’
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TAG
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1 2 3 7 8 94 5 6
Image acquisition Base calling
T G C T A C G A T …
Sequencing
Illumina Sequencing Technology
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Unravel20 wholetranscriptomes
In four days
Profile 200 gene expression samples
In less than two days
Analyzetwo human
methylomes
In one week
Sequence one cancer &one normal genome
At 30x coverage
What if, in one sequencing run you could…
OneSequencing
Run
SIMULTANEOUSLY
Run multiple applications requiring different read lengths
Whole genome sequencing
Targeted resequencing
Gene expression
Whole transcriptome
ChIP-seq
Metagenomics
De novo
Methylation
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Focused Genotyping
•HumanCVD BeadChip•BovineSNP50 BeadChip•CanineSNP20 BeadChip•CanineHD BeadChip•EquineSNP50 BeadChip•OvineSNP50 BeadChip•PorcineSNP60 BeadChip•MaizeSNP50 BeadChip•iSelect Custom Panel
Infinium
BeadChips
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Custom Genotyping
Infinium iSelect HD
• Interrogate virtually any SNP for any species• 24-sample format (3,000 to 68,000 attempted bead types)• 12-sample format (68,001 to 200,000 attempted bead types)
Custom GoldenGate Genotyping Panels
• Create assays tailored directly to their specific needs for targeted region genotyping or fine-mapping of candidate disease association regions.
• Interrogate 96, or from 384 to 1,536, SNP loci simultaneously
GoldenGate Indexing v1 Kit
• Screen up to 16 times as many samples per reaction as the standard GoldenGate Assay• Increase throughput from 288 samples per day to greater than 2000, while decreasing total reagent consumption. • Current plexity ranges for GoldenGate Indexing include 96-plex, 192-plex, and 384-plex. • The GoldenGate Indexing Assay is conveniently processed on the Universal-32 BeadChip
The Rosaceae – Fruits, Nuts and Flowers
Rosaceae BeadChips• Roscaeae BeadChip
– SA and NZ working in collaboration with RosBREED
– Using around 9,000 SNPs per species– Apple, Peach, Cherry, Strawberry– SNPs are from founders of major
breeding programs and novel cultivars– Optimised for cultivated populations
as opposed to wild crosses– Available March 2011
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Customize HiScanSQ To Meet Your Needs – Now or Later
SPLs: Samples HumanOmniExpress 12 sample BeadChips. Throughput varies by BeadChipFor illustration purposes only – consult your rep to discuss your expected sample throughput.Assumptions:6 Gb/day sequencing output. Manual sample processing: 1 HiScan, 0 Autoloader2.x, 0 Tecan liquid handling robots, 5 day work week; 2 FTEs. Automated sample processing: 5 day work week; 24 hour scanning powered by 1 AutoLoader2.x, 1 Tecan liquid handling robot.
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Predictions
Cost per data point will continue dramatic reduction for both Arrays and Sequencing
Sequencing output will scale quickly
Array content and complexity will increase significantly
Sample numbers for projects will grow
Sequencing and array solutions will continue to co-exist and complement
Cost, Complexity and Complementary Solutions
ARC Biotech Platform ImplementationThe First Genomes
• First genome sequencing: March 2011• Apple, pear, aphid, tick, amaranth, heartwater, avian
paramyxovirus• (15 apple genomes resequenced in 2010)
• First major genotyping experiments, from May 2011• Apple (RosBREED - USA)• Peach (ARC)• Cattle (ARC)• Sheep (US, ARC)
Agric-Biotech Smart Tools• Rapid characterization of novel genomes (plants,
animals, pathogens)• Gene expression and genotyping technologies• Molecular Breeding for rapid crop and animal
improvement: – Genome-wide selection– Marker assisted selection
• Tissue Culture and micro-propagation of plants• Cis-genesis and Genome Engineering• Reproductive biotechnologies• Vaccines, therapeutic proteins, diagnostics
Impact on Agricultural Research and Development
• Faster, better, more selective and more sensitive:– Plant Breeding– Animal Breeding– Cultivar identification (PBR)– Pathogen identification– Diagnostics– Vaccine development– Vaccine quality control and safety
• Bringing better crops and animals to farmer at all scales• Bringing novel, cash generating, sustainable products to
the market• Increasing food and income security
ARC: BP – the 5 year horizon
• By 2015, The ARC Biotech Centre will be a vibrant Agri-Biotech institution – a Centre of Excellence in research, training and services with National and International standing
• Regional integration of services and research• Training of new generation of researchers and
postgraduates• Implementation of high throughput systems for applied
and basic research• Increased funding and collaborative opportunities
