Plant Breeding and Agri Genomics
Team Genotypic 24 November 2012
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
10 PhDs and 78 MSc MTech BTech
Genotypic Family: The Best Genomics Experts Under One Roof
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
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 1998.
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)
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Certifications
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
DSIR Certified
R&D laboratory
What is Genomics? Simplified
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
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
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
What is Genomics? Simplified
Apply Arrays. Array technology is stable and best suited for large scale projects
“Un supported Methods”
Interpretation
Novel Designs
Standard protocols modified for novel applications
1000s of RNA /DNA extractions, 100s of arrays Guaranteed high quality data
Arrays are used for validation of SNPs, gene expression, gene copy changesPresented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Special from Genotypic
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
Million oligos made in one go.. Any Sequence.. Any application..
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Magical but True
NEXT GENERATION SEQUENCING
Dideoxy Sequencing
PCR
qPCR
Microarrays
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Parallel sequencing reactions on a microarray slide!
1 Million to 100 Million tiny wells (454 and Ion Proton)
500 Million spots- Illumina /SOLiD
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
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.
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
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.
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Next Generation Sequencing
Sequence reads with base quality, FASTQ format
Sequence ID
Quality ID
Sequence
Quality value for each base in ascii format
Fig1: Snapshot of the FASTQ file
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
At Genotypic = GAIIx illumina sequencer (2 machines)
150 bases Forward and 150 bases reverse
Ganesha
Jumbo
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Jumbo
SOLiD 5500xl- Wild fire
Lowest price per read >250 M readsChIP seqSmall RNAseqCounting applications:Gene Expression by NGSTomato sized genomes:Applications accuracy
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Ideal for amplicon sequencing, genotyping by sequencing and
RECONFIRMATION
From 10MB to 1GB data per run
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
80 Million reads per 4 hr run100 to 400 base single reads
The Magical Ion Proton sequencer arrived at Genotypic
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
> 50 Plants sequenced at Genotypic – transcriptomes, small RNA and resequencing trait associated genes and RRL
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
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
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
The chickpea Arrays are made in collaboration between
Genotypic and NIPGR and
available from Genotypic
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
De novo Chickpea Transcriptome
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 non-model plant with potent anti-diabetic properties
Krishna Prasad (VP Genomics Applications @ Genotypic)
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Examples of R&D projects
High outputs >30M reads per laneSOLiD 4GAIIxPollonator/IBS
Massive outputs >100M short readsSOLiD 5500xlHiSeq
Massive outputs 100M Long readsIon Proton
100MB to 1GB outputsQuick runs – validationsMiSeqIon PGM454Jr, GnuBio, IBS
Looong read technologies454 (500 bp to 1 KB)PacBio (10KB)Nanopores >10 KB
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
NGS current scenario Simplified
Long / medium PCR-> Fragment Library prep
Amplicon Seq (Seq primer tails)AmpliSeq (remove primer)Fluidim and RainDance PCR Prep methods
Massive targeting >5MBSureSelectArray capture (flexible)
Quick Targeting / indepth16S primer comboShort PCRsMetagenomics /Tilling aplications
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
NGS Targeted Sequencing Simplified
It is no more brute force 454+HiSeq
Generate Massive Data (100X to 200X)Hiseq 100 PE150 PE illumina (longer inserts)Ion Data 200 to 400 SE
ScaffoldingMate paid LibraryRRLs (replacement to BACS and Lambda)
Looong read technologies454 (500 bp to 1 KB)PacBio (10KB)
Validation of assemblyMicroarraysTranscriptomesDefining New Paradigms for Sequence Assembly
with Opgen. (optical Mapping – restriction mapping)
Whole Genome denovo Sequencing Simplified
Bioinformatics and what is expectedDifferent assembly alignment methodsManual verificationsReference sequences
Good old MicroarraysValidate junctions, low coverageORF orientation
Alternate NGS HiSeq by IonSOLiD by GAIIx
Sanger SequencingA few important genes
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Validating NGS Simplified and Making use of NGS
Genomics is high throughput Molecular biology
Plan which platform and combinationsGoal oriented plansFlexibility – based on resultsPilots / simulation studies
Analyze with a purposeIt is not software not bioinformaticsIt 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
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Applying Genomics- Simplified
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”
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
SNP discovery Challenges
From Discovery to SNP assays
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
Workflow
ChromosomeGenes
Sequence cDNA from tissue(s) of interest
SNPs only from transcribed regions– high value functional SNPsDisadvantage- non uniform spread across chromosomes
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
SNP discovery from cDNA (transcriptome) sequencing
ChromosomeSequencedregions
Increase coverage based on the requirement is possible
Reduce the representation uniformly throughout the genome
By choosing methylation sensitive restriction enzymes and at non repeat regions and regions that matter (in and around genic regions)
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Reduced Representation sequencing
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
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.
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
SNP discover options
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
DYN Israel
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
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.
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Summary
Thank you
&
Hope to work with you soon
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Genomics: Next Generation DNA Sequencing (NGS) and MicroarrayLargest Group in Linkedin – managed by Genotypic ~14000 userswww.linkedin.com/groups?about=&gid=1907871
Plant breeding & Genetics AND Agri Genomics >2500 userswww.linkedin.com/groups?mostPopular=&gid=3186656
Updates from genotypic are posted atwww.facebook.com/genotypic
We are closely connected with R&D units and support units of Genomics Tech companiesIon community, SOLiD community, NGS analysis user groups, Illumina webinars………USERs
Presented in Open house discussion on Plant genotyping and Agrigenomics organized by Genotypic, Bangalore on 24th Nov 2012
Updates and Networking