The History of DNA Sequencing Technology

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

Dr Jasper Rees ReesJ@arc.agric.za 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 ReesJ@arc.agric.za

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 Proteomics Databases Annotation Assembly Expression Analysis Bioinformatics Tissue Culture Construct development Transformation Regeneration Plant Transformation

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

Platforms and Programs Drought Tolerant Sorghum Apple Breeding Vaccine Development Animal Reproductive Biotechnologies Diagnostics Genomics 1 2 Proteomics Bioinformatics Plant Transformation 3 Functional Genomics Genome Engineering 4 Imaging NanoBiotechnology Protein Expression

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 SNP applications Genomes, De novo and re-sequencing SNPs and CNV calling Transcriptomes, SNPs and splicing variation Expression profiling Small RNA discovery and expression analysis GWAS Association Genetics SNP validation Candidate Genes Diversity studies

Platform Synergy for Agrigenomics Research Discovery Focused Research/Validation Screening >109 108 107 106 105 104 103 102 10 1 Whole Genome Single Marker Discover Interrogate Validate/Screen Platform HiSeq 2000 iScan/HiScan SQ BeadXpress ► De novo Genome Sequencing mRNA Sequencing & Gene Expression SNP Discovery DNA Methylation, Targeted Resequencing ► Genome-wide marker / phenotype association Focused marker / phenotype association Genetic Identity Confirmation QTL Mapping ► Genetic Purity Screening Illumina is a market leading company providing tools for genetic analysis – keen to ensure develop and provide correct tools to support Agrigenomics research Genome wide discovery studies supported by the the GA, genome wide validation and association studies can be carried out on the iScan where more than one million markers can be assessed per sample in a single assay. The iScan is also suited to conduct more focussed studies and screening applications in lower plex assays. The BX platform is geared to screening studies in which high sample throughput rates are required with lower assay multiplexing. Thus Illumina products cover a wide range of genotyping expt designs right from whole genome sequencing through to low multiplex genotyping. ► Parentage / Lineage Applications ► Marker Assisted Selection ► Disease Screening

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

Illumina Sequencing Technology 5’ 3’ G T C A DNA (0.1-1.0 ug) T G C A G T Sample preparation Cluster growth Single molecule array [Enter any extra notes here; leave the item ID line at the bottom] Avitage Item ID: {{C2A4BBD5-93E1-4B59-A5D0-E38C126BE098}} Sequencing 1 2 3 7 8 9 4 5 6 Base calling T G C T A C G A T … Image acquisition

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 Unravel 20 whole transcriptomes In four days Analyze two human methylomes In one week Whole genome sequencing Profile 200 gene expression samples In less than two days One Sequencing Run Targeted resequencing Gene expression Methylation The goal of this slide is to capture the power and scope of research that will be made possible with HiSeq 2000 De novo Metagenomics ChIP-seq Whole transcriptome

Focused Genotyping Infinium BeadChips HumanCVD BeadChip BovineSNP50 BeadChip CanineSNP20 BeadChip CanineHD BeadChip EquineSNP50 BeadChip OvineSNP50 BeadChip PorcineSNP60 BeadChip MaizeSNP50 BeadChip iSelect Custom Panel

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 Maize is $195 per sample

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.

Predictions Cost, Complexity and Complementary Solutions 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

ARC Biotech Platform Implementation The 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

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