Plant Breeding and Agri Genomics - Genotypic Technology of Genotypic... · Plant Breeding and Agri Genomics ... Bangalore on 24th Nov 2012. ... to work together with seed companies

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


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)


Certifications


DSIR Certified

R&D laboratory

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


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..


Magical but True

NEXT GENERATION SEQUENCING

Dideoxy Sequencing

PCR

qPCR

Microarrays


Parallel sequencing reactions on a microarray slide!

1 Million to 100 Million tiny wells (454 and Ion Proton)

500 Million spots- Illumina /SOLiD


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.


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.


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


At Genotypic = GAIIx illumina sequencer (2 machines)

150 bases Forward and 150 bases reverse

Ganesha

Jumbo


Jumbo

SOLiD 5500xl- Wild fire

Lowest price per read >250 M readsChIP seqSmall RNAseqCounting applications:Gene Expression by NGSTomato sized genomes:Applications accuracy


Ideal for amplicon sequencing, genotyping by sequencing and

RECONFIRMATION

From 10MB to 1GB data per run


http://find.lifetechnologies.com/ionfirstsix/fl

http://www.iontorrent.com/applications-speed/

80 Million reads per 4 hr run100 to 400 base single reads

The Magical Ion Proton sequencer arrived at Genotypic


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


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


The chickpea Arrays are made in collaboration between

Genotypic and NIPGR and

available from Genotypic


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)


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


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


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


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


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”


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


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)


Reduced Representation sequencing


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.


SNP discover options


DYN Israel






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.


Summary

Thank you

&

Hope to work with you soon


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


Updates and Networking

Documents

Plant Breeding and Agri Genomics - Genotypic Technology of Genotypic... · Plant Breeding and Agri Genomics ... Bangalore on 24th Nov 2012. ... to work together with seed companies