Plant Breeding and Agri Genomics. Team Genotypic 24 November 2012

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1 Plant Breeding and Agri Genomics Team Genotypic 24 November 2012

2 Genotypic Family: The Best Genomics Experts Under One Roof 10 PhDs and 78 MSc MTech BTech

3 ABOUT US! Genotypic is a Genomics company, which has consciously focused on building its capability in adaptation of multiple genomic technologies and applying the same to challenging questions posed by users across the life sciences spectrum. Genotypic is India s first Genomics Company established in Genotypic is an ISO 9001:2008 Accredited Company. Genotypic s lliance Partners are in Australia, Israel, Singapore, UK, Germany, South Africa Malaysia, Canada, and USA. Successfully completed >500 NGS projects and >2000 microarray projects. Users >400 in India> 300 worldwide AgriGenomics, SNP Genotyping services DYN R&D Israel Rapid SNP Discovery (functional and genome wide)

4 Certifications DSIR Certified R&D laboratory

5 What is Genomics? Simplified

6 What is Genomics? Simplified Magical High throughput Molecular biology An Enabling Technology Has become THE WAY of Biological Research 100 Million sequence reads 1 Million oligos arrayed of any sequence 1000s of PCRs in nano liter volumes. Ultra Low cost per Base

7 Special from Genotypic Apply Arrays. Array technology is stable and best suited for large scale projects 1000s of RNA /DNA extractions, 100s of arrays Guaranteed high quality data Standard protocols modified for novel applications Novel Designs Un supported Methods Interpretation Arrays are used for validation of SNPs, gene expression, gene copy changes

8 Magical but True Million oligos made in one go.. Any Sequence.. Any application.. Customs Designs By Genotypic 8 Samples 10K to 100K SNPs is possible Application No. Of Designs CGH 55 COC / CH3 128 GXP 230 Targeted capture (OAC, Insolution) SureSelect, haloplex, AmpliSeq) 75 Total >500 Major Taxonomy Class No. Of Designs Bacterial 122 Mammal 176 Plant 55 Other (including metagenomics) 100 Total >500

9 NEXT GENERATION SEQUENCING Dideoxy Sequencing PCR qpcr Microarrays

10 Parallel sequencing reactions on a microarray slide! 1 Million to 100 Million tiny wells (454 and Ion Proton) 500 Million spots- Illumina /SOLiD

11 NGS is changing biology with numerous applications Anything that is living, lived are being sequenced worldwide Like PCR, NGS is being applied in all domains of biology and is becoming an essential tool.

12 Next Generation Sequencing Sequencing of 100s of bacterial genome in a day Sequence a few selected regions of human genome in a week For 1000s of individuals Complete human genome sequencing in a DAY! Plant transcriptomes in a week Plant Genomics Simplified: Marker discovery, Back crossing.

13 Sequence reads with base quality, FASTQ format Sequence ID Sequence Quality ID Quality value for each base in ascii format Fig1: Snapshot of the FASTQ file

14 At Genotypic = GAIIx illumina sequencer (2 machines) 150 bases Forward and 150 bases reverse Ganesha Jumbo Jumbo

15 SOLiD 5500xl- Wild fire Lowest price per read >250 M reads ChIP seq Small RNAseq Counting applications: Gene Expression by NGS Tomato sized genomes: Applications accuracy

16 Ideal for amplicon sequencing, genotyping by sequencing and RECONFIRMATION From 10MB to 1GB data per run

17 The Magical Ion Proton sequencer arrived at Genotypic 80 Million reads per 4 hr run 100 to 400 base single reads

18 > 50 Plants sequenced at Genotypic transcriptomes, small RNA and resequencing trait associated genes and RRL

19 Genotypic works with Plants animals virus bacteria fungi. A variety of novel applications Amplicon sequencing deep sequencing from 100s of samples POOLED Capture and sequencing 100s of genes / regions in 100s of pooled samples High yield low yield Resistant sensitive Bulk segregrant analysis Tilling by sequencing

20 De novo Chickpea Transcriptome The chickpea Arrays are made in collaboration between Genotypic and NIPGR and available from Genotypic

21 Examples of R&D projects Publications from Genotypic De novo transcriptome assembly (NGS) of Curcuma longa L. rhizome reveals novel transcripts related to anticancer and antimalarial terpenoids. Next Generation Sequencing and de novo transcriptome analysis of Costus pictus D. Don, a nonmodel plant with potent anti-diabetic properties Krishna Prasad (VP Genomics Genotypic)

22 NGS current scenario Simplified High outputs >30M reads per lane SOLiD 4 GAIIx Pollonator/IBS Looong read technologies 454 (500 bp to 1 KB) PacBio (10KB) Nanopores >10 KB Massive outputs >100M short reads SOLiD 5500xl HiSeq Massive outputs 100M Long reads Ion Proton 100MB to 1GB outputs Quick runs validations MiSeq Ion PGM 454Jr, GnuBio, IBS

23 NGS Targeted Sequencing Simplified Long / medium PCR -> Fragment Library prep Amplicon Seq (Seq primer tails) AmpliSeq (remove primer) Fluidim and RainDance PCR Prep methods Massive targeting >5MB SureSelect Array capture (flexible) Quick Targeting / indepth 16S primer combo Short PCRs Metagenomics / Tilling aplications

24 Whole Genome denovo Sequencing Simplified Generate Massive Data (100X to 200X) Hiseq 100 PE 150 PE illumina (longer inserts) Ion Data 200 to 400 SE It is no more brute force 454+HiSeq Scaffolding Mate paid Library RRLs (replacement to BACS and Lambda) Looong read technologies 454 (500 bp to 1 KB) PacBio (10KB) Defining New Paradigms for Sequence Assembly with Opgen. (optical Mapping restriction mapping) Validation of assembly Microarrays Transcriptomes

25 Validating NGS Simplified and Making use of NGS Bioinformatics and what is expected Different assembly alignment methods Manual verifications Reference sequences Good old Microarrays Validate junctions, low coverage ORF orientation Alternate NGS HiSeq by Ion SOLiD by GAIIx Sanger Sequencing A few important genes

26 Applying Genomics- Simplified Genomics is high throughput Molecular biology Plan which platform and combinations Goal oriented plans Flexibility based on results Pilots / simulation studies Analyze with a purpose It is not software not bioinformatics It is analyzing HT Mol Bio data!! Decisions, Concepts, Products, IP The most important part - this is not just bioinformatics- - It is biology oriented Genome informatics

27 SNP discovery Challenges Plant Genomes are large and Expensive to sequence as well as analyze Exception 1 Arabidopsis small genome and can be sequenced in one lane Exception 2 It is now possible to sequence 1GB sized genomes at low cost. Wish 1 Validated SNPs Wish 2 Valuable SNPs

28 From Discovery to SNP assays Workflow Discovery of SNPs Quality control and Identification of valuable SNPs Verify genotype-phenotype- if required in collaboration with others who want to use the markers Design test and Develop assays (DYN technology) Apply them in your breeding programs. (DYN kits / Genotypic s services) Agrigenomics consortium to work together with seed companies and academic scientists to generate focused sequence databases - as a base of applying these

29 SNP discovery from cdna (transcriptome) sequencing Chromosome Genes Sequence cdna from tissue(s) of interest SNPs only from transcribed regions high value functional SNPs Disadvantage- non uniform spread across chromosomes

30 Reduced Representation sequencing Chromosome Sequenced regions Reduce the representation uniformly throughout the genome Increase coverage based on the requirement is possible By choosing methylation sensitive restriction enzymes and at non repeat regions and regions that matter (in and around genic regions)

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32 SNP discover options 1. Sequencing of targeted regions (chromosomal or selected genes) 2. Restriction assisted sequencing (reduced representation sequencing methylation + or -) 3. Quick whole genome sequencing (now possible for <1 GB sized plants) transcriptome sequencing 4. Ultra high throughput sequencing of very small regions of interest in a pool of plants (100s of plants and a few regions) Tilling applications and trait associated marker discovery 5. Microarrays for mass scale validation of SNVs identified by NGS (filtering for high quality and useful SNPs) and other smart ways of using arrays in plant genomics.

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39 Summary Applications of SNP genotyping germplasm characterization and barcoding, parent and hybrid purity testing, seed lot testing, trait associated markers for marker assisted breeding, back crossing, early generation selection, linkage block analysis and selection, enrichment of complex F1s,Pyramiding of disease-resistant genes. NEW: Convert SSR markers to robust SNP genotyping assays Apply NGS to develop SNP markers for various applications in sequenced and unsequenced plants.

40 Thank you & Hope to work with you soon

41 Updates and Networking We are closely connected with R&D units and support units of Genomics Tech companies Ion community, SOLiD community, NGS analysis user groups, Illumina webinars USERs Genomics: Next Generation DNA Sequencing (NGS) and Microarray Largest Group in Linkedin managed by Genotypic ~14000 users Plant breeding & Genetics AND Agri Genomics >2500 users Updates from genotypic are posted at