2015. Pegadaraju Venkatramana. Array Tape Platform and its appliccation in genomics assisted breeding

Confidential M A K I N G O U R W O R L D A B E T T E R P L A C E 1

5th International Conference

On Next Generation Genomics and

Integrated Breeding for Crop Improvement

February, 18th 2015 ICRISAT, Patancheru, India

Douglas Scientific Array Tape™ Platform And It’s

Application In Genomics Assisted Breeding

Pegadaraju Venkatramana

Douglas Scientific

Ross Higgins

Venki

Dan Harms


Outline:

Introduction to Douglas Scientific

Array Tape™ Automation - Technology Overview

Array Tape Value proposition

IntelliQube- Next Generation Technology (qPCR and Endpoint for Medium to High Throughput Range)

Applications Related to Molecular Breeding

Q & A


Breeder’s Key Questions

Whole Genome /

Transcriptome Sequencing

SNP Discover & SNP Validation

—Assay performance

—Genome distribution

—PIC values

Fingerprinting

Germplasm

Forward

Breeding

Backcross

Breeding

- Hybrid/Varietal ID

- Inbred panel

- Trait Specific panel

- Pooled Seed Testing

QA/QC

Panel

- Foreground

- Background

- Recombinant

QTL GS

Model

GEBVs

• Characterization

- Pop Structure & Kinship

- Similarity Matrix

• Intellectual Property

Line Selection/Superior Alleles/Line selection & Development

Ph

ase I

Ph

ase II

Ph

ase III

Ph

ase IV

Genomics Assisted Selection

- MAS

- MARS

- F2-En..

GWAS Haplotypes

Model Building

NG

S & A

rray Base

d

Platfo

rms

Au

tom

ated SN

P G

en

otyp

ing

Platfo

rms

Discovery

Production


Strong History of Market Success in AgBio


Mission Statement

Douglas Scientific is dedicated to making our world a better place by delivering

innovative laboratory automation.


Array Tape is a Continuous Polymer Media


Array Tape Platform Configurations

Throughput

500,000 to 1 Million Data Points/Year

1 Million to 6 Million Data Points/Year


9,600 Data Points/Year



Throughput





The Nexar End-point PCR System

Array Tape® Nexar® Soellex® Araya®

Microplate Replacement

Liquid Handling

Thermal Cycling

Fluorescence Detection


Array Tape Platform Video


Samples Tested With Array Tape


General Information about Array Tape

Integrated inline processing

– Laboratory and labor efficiency

– Maximize throughput and turn-around times

Miniaturized reactions, typically 1.6uL

– Up to a 90% reduction in chemistry

What about the data quality?!


Miniaturization and Data Quality Array Tape Platform

1.6µL Reactions Real-Time PCR Instrument

5µL Reactions

100% concordance between platforms

100% accuracy with published results


Array Tape System Is Chemistry Independent Platform


Array Tape Technology Is Crude Compatible


Profound Results for an Actual Customer

Documented throughput gains of 10x

Cost reductions of up to 90%

Reagent Consumable DNA Prep Depreciation Repair/Maint Labor

Plate Array Tape

Microtiter Plates vs. Array Tape™ Agbio customer

– October 2010 - running 500,000 data points per year

– October 2014 – running 50,000,000 data points/year (3 technicians)

Clinical Diagnostics customer

– Nov 2013 – 15,000 data points/day, 8 ViiA7, 15 lab techs across 3 shifts

– October 2014 – 36,000 data points/day, 1 platform, 3 lab techs, with capacity left over for other tasks.

Capacity Validation

Conclusions Inline Automation using Array Tape Platform is a proven and efficient process for high

throughput genotyping

– Reduces labor costs

– Reduces floor space (indirect costs)

– Reduces capital costs

– Increases throughput

– Decreases time from sample to data

Data Quality and Accuracy

High quality data on Array Tape as opposed to plate based operation

Low Cost per Data Point

Significantly reduces cost per data point by miniaturizing the reaction volumes. ($1/sample with 100 SNP’s fingerprint)

Durable & Flexible Automation Technologies

Current life span is more than 5 years, to date none of the systems deployed since, 2009 have been decommissioned.

Access and Utilization of Laboratory Professionals

Running 40M data points/yr requires 3 lab techs

Informatics for Analysis

Intellics Software Suite



Throughput





The IntelliQube End-point PCR System

Array Tape® IntelliQube® IntelliCyclerTM IntelliQube®


Liquid Handling

Thermal Cycling






Liquid Handling

Thermal Cycling






Liquid Handling

Thermal Cycling






Liquid Handling

Thermal Cycling





Throughput





The IntelliQube Real-Time PCR System

Array Tape® IntelliQube® IntelliQube® IntelliQube®


Liquid Handling

Thermal Cycling






Liquid Handling

Thermal Cycling






Liquid Handling

Thermal Cycling






Liquid Handling

Thermal Cycling



IntelliQube System Highlights - Optics/Dyes

LEDs (470-627 nm)

Dye Channel 1 2 3 4 5

Excitation Filter (nm) 480 530 548 580 620

Emission Filter (nm) 510 565 580 625 705

JOETM NEDTM ROXTM

VIC®/HEXTM TAMRATM Cal Fluor® Red 610

Cal Fluor®Orange 560 Cal Fluor® Red 590 Texas Red®

FAMTMCy5®

Quasar® 670

Common Dyes

High resolution CCD camera

Filtered LED excitation source supports 5 dye channels

Multiplex data capture in 10 seconds or less

FAMTM, JOETM, VIC, HEXTM, NEDTM, TAMRATM and ROXTM are trademarks of Life Technologies Corporation and its subsidiaries. VIC® and Texas Red® are registered trademarks of Life Technologies Corporation and its subsidiaries. Cy5® is a registered trademark of GE Healthcare Bio-Sciences Corp. Cal Fluor® and Quasar® are registered trademarks of Biosearch Technologies, Inc.


IntelliQube System Highlights – Est. Throughput

qPCR (Inline)* with 768-Well Array Tape

Reactions 384-well plate Equivalents

Per 8 hour shift 4,600 12

Per year (1 shift/day, 250 days/year)

1.1 million 3,000

End-Point PCR with Waterbath with 384-Well Array Tape

Reactions 384-well plate Equivalents

Per 8 hour shift

23,000

60

Per year (1 shift/day, 250 days/year)

5.8 million 15,000

*Throughput is dependent on specific cycling conditions/chemistry. All estimates are based on a 73 minute TaqMan® SNP Genotyping assay protocol


IntelliQube System Highlights - qPCR Performance

Excellent Cq Uniformity 10K Copies gDNA

2 Fold Resolution 5K and 10K gDNA

8 Log Dynamic Range 10-fold serial dilution


Recent SNP Data

Sample Type:

– Crude prep corn

Assay:

– KASP Corn Assay

PCR:

– IntelliCycler


Thank you!

De novo sequencing of sunflower genome for SNP discovery using RAD (Restriction site Associated DNA) approach

Disease

Downy Mildew

Rust

Sclerotinia

Verticillium

Alternaria

Stem Cancer

Rhizopus

Insects

Sunflower Moth

Weevil

Midge

Longhorn Beetle

Wire Worms

Weeds

Biennials Wormwood

Foxtail

Kochia

Redroot Pigweed

Wild sunflower

Wild Buckwheat

Quality traits

High Oleic soybeans

Pegadaraju et al., BMC Genomics 2013

• Sunflower has a complex genome of 3.5Gb & remains to be sequenced

• Discovery of SNPs variants in sunflower would require development & assembly of large island of DNA sequence to detect SNPs • RAD Long Read technology (Floragenex) coupled with bioinformatics analysis was adapted to de novo assemble of sunflower genome & identify SNP markers

Helianthus Annuus Sequence Analysis Pipeline

Sequence Assembly & SNP discovery RAD –Seq Assembly Results & Repeat Element Contribution

Pegadaraju et al., BMC Genomics 2013

Sunflower Genotyping Panel

Advanta

USDA

Seeds

2000

Genosys

Mycogen

NuFlower

CHS

1291

samples

Diversity Panel Mapping Panel

HA89 x RHA464

F1

Self

139, F2 lines genotyped

8723

NSA Sunflower SNP

Chip

Mayo Agro

B-line x RHA 464 CR 29 x RHA 468

F1

Self


F1

Self


RAD Sequencing

Linkage Mapping

Breeding Panel

Array Based Genotyping

De novo Sequencing

16,464 SNP’s 8723 SNP’s 384 SNP’s

Optimal Marker Panel for Routine Breeding


Molecular Breeding Application on Array Tape Platform

Molecular Breeding:

• QTL Mapping

• Markers assisted breeding

• Marker assisted backcrossing

• Genomic Selection Production

QA/QC:

•Hybrid purity

•PVP

•Variety verification


parental Downy Mildew

Downy

Mildew Downy Mildew

Downy

Mildew

Downy

Mildew

Downy

Mildew

Lot/Row phenotype NSA_007226

C2

NSA_00

4663

NSA_00406

0

NSA_004

822

NSA_00307

9

NSA_003

689

1064-1 S S S S S S S

1064-2 S S S S S S S

1671-1 S S S S S S S

1671-2 S S S S S S S

1671-3 S S Und. S S S S

1671-4 S S S S S S S

1671-5 S S S S S S S

1672-1 S S S S S S S

1672-2 S S S S S S S

1672-3 S S S S S S S

1672-4 S S S S S S S

1672-5 S S S S S S S

1673-1 S S S S S S S

1673-2 S S S S Und. S S

1673-3 S S S S S S S

1673-4 S S S S S S S

1673-5 S S S S S S S

1841-1 S S und. S S S S

1841-2 S S und. und. und. und. S

1937-1 S S S S S S S

1937-2 S S S S Und. S S

3104-1 R R S R R R R

3104-2 R R?? S R R R R

3104-3 R R S R R R R

3105-1 R R S R R R R

3105-2 R R S R R R R

3105-3 R R?? S R Und. R R

3143-1 R S S R Und. R S

3143-2 R S S R S R S

3143-3 R R?? und. und. und. und. und.

3144-1 R R?? R R S R S

3144-2 R R?? R R S R S

3144-3 R R?? und. R S R S

3145-1 R R?? R R R R Und.

3145-2 R R R R R R R

3145-3 R R?? R R R R Und.

3146-1 R R?? R R R R Und.

3146-2 R R?? R R R Und. R

3146-3 R R?? R R R R Und.

3147-1 R R S R R R R

M_001726 28.398

ORS716 28.659

M_001104 28.827

M_002138 M_002319 29.031

M_001953 29.081

M_000532 M_000930 M_001209 29.242

M_001441 29.396

M_000844 29.407

M_001174 29.408

M_002515 29.415

M_000077 M_000083 M_000166 M_000167 M_000174

M_000258 M_000321 M_000330 M_000356 M_000376

M_000426 M_000511 M_000548 M_000600 M_000630

M_000639 M_000657 M_000661 M_000701 M_000802

M_000836 M_000840 M_000850 M_000857 M_000868

M_000869 M_000883 M_000898 M_000914 M_000915

M_000921 M_000931 M_001051 M_001121 M_001128

M_001162 M_001216 M_001225 M_001239 M_001249

M_001274 M_001278 M_001282 M_001342 M_001457

M_001464 M_001515 M_001527 M_001537 M_001605

M_001627 M_001644 M_001650 M_001674 M_001681

M_001767 M_001813 M_001834 M_001880 M_001910

M_001965 M_002003 M_002015 M_002016 M_002074

M_002253 M_002264 M_002326 M_002327 M_002387

M_002421 M_002437 M_002468 M_002552 M_002705

M_002722 M_002736 M_002796

29.434

PlARG 29.435

M_002615 29.436

M_001434 29.449

M_001230 29.565

M_002498 29.588

CRT277-1 29.602

M_001139 29.646

M_002088 29.650

M_000254 M_002657 29.660

M_002333 ORS1182 29.663

M_001081 M_002571 29.664

M_002589 29.665

ORS1128 29.666

HT353 ORS543 M_000024 M_000035 M_000037

M_000055 M_000127 M_000129 M_000145 M_000149

M_000182 M_000228 M_000259 M_000264 M_000283

M_000311 M_000322 M_000351 M_000370 M_000395

M_000398 M_000429 M_000433 M_000435 M_000441

M_000458 M_000462 M_000467 M_000471 M_000483

M_000496 M_000526 M_000536 M_000541 M_000573

M_000582 M_000611 M_000614 M_000617 M_000633

M_000643 M_000681 M_000690 M_000704 M_000708

M_000728 M_000741 M_000768 M_000771 M_000782

M_000792 M_000793 M_000854 M_000879 M_000889

M_000894 M_000895 M_000897 M_000927 M_000941

M_000955 M_000963 M_000970 M_000986 M_000987

M_000995 M_000997 M_001001 M_001012 M_001043

M_001057 M_001077 M_001091 M_001132 M_001134

M_001138 M_001151 M_001154 M_001158 M_001170

M_001204 M_001221 M_001232 M_001265 M_001284

M_001288 M_001303 M_001345 M_001375 M_001398

M_001406 M_001416 M_001433 M_001440 M_001442

M_001469 M_001470 M_001479 M_001485 M_001510

M_001529 M_001564 M_001579 M_001611 M_001615

M_001618 M_001646 M_001654 M_001655 M_001658

M_001667 M_001701 M_001755 M_001765 M_001867

M_001901 M_001922 M_001925 M_001950 M_001983

M_001989 M_002009 M_002012 M_002019 M_002041

M_002042 M_002062 M_002161 M_002162 M_002168

M_002194 M_002196 M_002203 M_002204 M_002220

M_002238 M_002265 M_002273 M_002283 M_002329

M_002362 M_002366 M_002379 M_002395 M_002419

M_002433 M_002434 M_002442 M_002462 M_002465

M_002466 M_002481 M_002484 M_002509 M_002525

M_002564 M_002575 M_002666 M_002673 M_002680

M_002684 M_002690 M_002708 M_002713 M_002714

M_002727 M_002732 M_002743 M_002756 M_002767

M_002768 M_002795

29.670

M_001191 29.703

M_002706 29.770

M_002717 29.795

ORS610 29.858

CRT394 29.929

0.24 cM

(265 markers)

LG1

RHA 464 (PlARG)

Fine Mapping of Sunflower Downy Mildew

Resistance Genes, PlARG



Concept Validation of a Pooled Seed

Testing Method on Array Tape Platform

Cultivated Sunflower Wild Sunflower

—Self compatible

—Unbranched growth

—Single large head

—Reduced germination dormancy

—Self-In compatible

—Many branches

— Seed germination dormancy


Screening the target SNP on a expanded panel

of sunflower lines

Cultivated

control

Wild control Intermediate

F1 hybrids

Cultivated

Wild

Genotypes Total

WT

allele

Cultivated

allele Both

Cultivated

lines 30 0 30 0

Wild

individuals 193 165 0 28


Testing the limit of detection for the

assay

1:100

1:1000 1:3000

Negative

Controls

1:5000

1:500

Negative

Controls

1:200

1:2000

1:500 1:1000

1:100

DNA Spike Seed Spike


Validation of Bonafide BDI™ – WildSUN

on spiked samples

Sample

sample

call

Computed % in

sample

actual

%

difference

(%)

Upper bound of

True % Impurity

R3 sample 1 1 of 10 0.02 0.02 0.00 0.10

R3 sample 2 2 of 10 0.04 0.04 0.00 0.14

R3 sample 3 3 of 10 0.07 0.06 0.01 0.19

R3 sample 4 3 of 10 0.07 0.10 -0.03 0.19

R3 sample 5 0 of 10 0.00 0.00 0.00 0.06

R3 sample 6 8 of 10 0.32 0.20 0.12 0.66

R3 sample 7 5 of 10 0.14 0.14 0.00 0.30

R3 sample 8 0 of 10 0.00 0.00 0.00 0.06

R3 sample 9 8 of 10 0.32 0.50 -0.18 0.66

R3 sample 10 8 of 10 0.32 0.30 0.02 0.66

R3 control 0 of 10 0.00 0.00 0.00 0.06


Cross Validation of Bonafide BDI™ –

WildSUN Test on Separate Platforms

ABI Douglas

DNA amt. 10 ng/ul sub sample 5 ng/ul sub sample

Label Call call rate pos. sample? Call call rate pos. sample?

Sample 1a Neg. N.C.

Sample 1b Neg. Neg.

Sample 1c Neg. 0 of 3 N Neg. 0 of 2 N

Sample 2a N.C. Neg.

Sample 2b Neg. Neg.

Sample 2c Neg. 0 of 2 N Neg. 3 of 3 N

Sample 3a Pos. Neg.

Sample 3b Pos. Pos.

Sample 3c Pos. 3 of 3 Y Pos. 2 of 3 Y

Sample 4a Pos. Pos.

Sample 4b Pos. Pos.


Sample 5a Pos. Pos.

Sample 5b Pos. Pos.


Blank Neg. Neg.

F1 Wild Pos. Pos.

F1 Wild Pos. Pos.

Cultivated Neg. Neg.

Cultivated Neg. Neg.


Genetic Purity/ Varietal ID Testing

Key Goal: To determine all individuals of a line/lot/bag are

constituted of same genetic make up

Seed produces / regulators use genetic purity testing to

identify the below:

— % Outcrosses

— % Self's

— % Seed mixes

— % Seed swaps

Key challenge:

– Current methods are conventional and testing is

conducted with very limited genetic markers

– Methods don’t work effectively in vegetables


SNP Based Genetic Purity Testing

• Key Features

—Detects single nucleotides changes

—Co-dominant in nature

— Highly abundant in number

—Multiplexed

Features DNA Test GOT

Accuracy High Low

Environmental

Influence No Yes

Sample Size 3000 seeds

400 seedlings Time Required 2 days 90-120 days

Cost effective Yes No

Seed

germination Not required Required

Seed grinding Required Not Required

Key Requirements:

—Uniform genome distribution

—Polymorphic

—Minimal number (Cost effective)

—Robust assay performance —Generic set of industry wide usage in various crops


Questions