The History of DNA Sequencing Technology

1. The History of DNA Sequencing Technology

2. ARC Biotechnology Platform Dr Jasper Rees ReesJ@arc.agric.za

3. ARC Biotechnology PlatformIn 2010, the ARC set out to create a new single integrated platform for advanced high throughput, next generation technologies Dr Jasper Rees ReesJ@arc.agric.za

4. Vision To create a world class biotechnology platform to lead research in agriculture in Africa

5. 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

6. 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

7. Service and Research Facilities Model 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 Unit Each sub-platform to have a development group responsible for introducing new technology and applications into the core services 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 Development group Research teams within platform Research teams at Institutes 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

8. Biotech Platform Phase I – 2010/11 Genomics Proteomics Bioinformatics Plant Transformation DNA Isolation Next Gen Sequencing Genotyping Genetic Mapping Sample Preparation 2D PAGE MALDI-MS LC/GC-MS-MS Databases Annotation Assembly Expression Analysis Tissue Culture Construct development Transformation Regeneration Development group Development group Development group Development group Research teams within platform Research teams within platform Research teams within platform Research teams within platform

9. Biotech Platform Phase II/III: 2012-2015 Marker Assisted Breeding Plant Phenotyping Genome Engineering Functional Genomics Robotic platform High throughput imaging and sensing GC MS/MS Informatics Reverse Genetics Gene identification Vector Development Construct development Robotic platform High throughput DNA Isolation Genotyping Informatics Mutation Breeding Gene Silencing Zinc Finger Nucleases Gene targeting Male Sterile Technology Targeted Mutagenesis

10. Platforms and Programs

11. 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

12. 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 • BonganiNdimba: Proteomics: Cereals and Fruit • DamarisOdeny; Genetics, Genomics: Vegetables • Joseph Mafofo: Genomics, plant pathogens: Fruit • Dirk Swanevelder: Genomics: Plant Pests, Cereals • FaraiMuchadeyi: Genotyping: Animal Genetics

13. Genomics Applications Sequencing applications SNP applications GWAS Association Genetics SNP validation Candidate Genes Diversity studies • 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

14. Platform Synergy for Agrigenomics Research Screening Discovery Focused Research/Validation >109 108 107 106 105 104 103 102 1 10 Single Marker Whole Genome Discover Interrogate Validate/Screen Platform HiSeq 2000 iScan/HiScan SQ BeadXpress ► Genetic Purity Screening ► Parentage / Lineage Applications ► Marker Assisted Selection ► Disease Screening

15. IlluminaHiScanSQ 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

16. 3’ 5’ G C T A C G T A C T A C C 5’ C C G A T A A A C G T T T A T G G G C 1 2 3 4 5 6 7 8 9 Base calling TGCTACGAT … Illumina Sequencing Technology DNA(0.1-1.0 ug) Sample preparation Single molecule array Cluster growth Sequencing Image acquisition

17. What if, in one sequencing run you could… • Sequence one cancer &one normal genome • At 30x coverage SIMULTANEOUSLY Run multiple applications requiring different read lengths Unravel20 wholetranscriptomes In four days Whole genome sequencing Analyzetwo humanmethylomes In one week OneSequencingRun Targeted resequencing Gene expression Methylation De novo Metagenomics Profile 200 gene expression samples In less than two days ChIP-seq Whole transcriptome

18. Focused Genotyping

19. Custom Genotyping

20. The Rosaceae – Fruits, Nuts and Flowers

21. RosaceaeBeadChips • RoscaeaeBeadChip • SA and NZ working in collaboration withRosBREED • 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

22. Customize HiScanSQ To Meet Your Needs – Now or Later SPLs: Samples HumanOmniExpress 12 sample BeadChips. Throughput varies by BeadChip For 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.

23. 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

24. 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)

25. 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

26. 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

27. 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

28. Thank you Dr Jasper Rees ReesJ@arc.agric.za