Semiconductor Sequencing Applications for Plant Sciences

The content provided herein may relate to products that have not been officially released and is subject to change without notice.

Ion PGM™ & Ion Proton™ Systems and Applications November 2012

Life Technologies™ | 1

Visit the Life Technologies website for more information.

http://www.lifetechnologies.com/us/en/home.html?CID=slideshare-plant-sequencing-webinar-12112012


2


Ion Torrent

Founded in 2007 by Jonathan Rothberg

− Pioneered next gen sequencing

− Founder of 454, CuraGen, Raindance

Acquired by Life Technologies in Jul 2010

Over 250 chemists, molecular biologists, engineers, software developers and bioinformaticists across 5 R&D sites

First PostLight™ sequencing technology launched in Dec 2010 (Ion PGM™ System)

First Postlight™ genome-scale sequencer shipped in Sep 2012 (Ion Proton™ System)

MA

CA CT

TX


Direct Detection of Hydrogen Ions and Conversion to an Electrical Signal Read by a Transistor Substrate

dNTP

H+

∆ pH

∆ Q

Sensing Layer

Sensor Plate

∆ V

Bulk Drain Source

Silicon Substrate To column

receiver

Rothberg J.M. et al Nature doi:10.1038/nature10242



Ion Semiconductor Sequencing Systems:

PGM™ for Genes. Proton™ for Genomes. Sequencing for All.

For more information, visit lifetechnologies.com/ionsequencing


http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Sequencing/Semiconductor-Sequencing.html?CID=fl-ionsequencing


Ion PGM™ Chip Ion User Q20 bases

314 Rmease 143,516,106

316 Corebotz 786,016,516

318 Wtr 1,275,271,258

Ion PGM™ and OneTouch™ 2 Systems with 3-Series Chips

Ion 318™ Chip

Ion 316™ Chip

Ion 314™ Chip

External Performance Well Beyond Spec (Ion Community RecogntION Runs)



Ion Proton™ System Bringing Rapid Genome-Scale Sequencing to Every Lab

Runs all Ion P-Series chips

Benchtop system containing state-of-the-art electronics to support the highest throughput

− Dual 8-core Intel® Xeon® Sandy Bridge processors

− 128 GB of RAM

− Dual Altera® Stratix V FPGAs

− NVIDIA® Tesla® C2075 GPU

− 11 TB of hybrid (SSD&HDD) storage

− Ubuntu® 11.10 operating system


hnolog1es lrwllrogen A.!:lpt edBto,pte m G1bc0 Mott'NI.OrPiobH" Novu T qM:III"" Amboon !onTOO"rent


Single Day Workflow with the Fastest Sequencing Runs

ION LIBRARY

KITS

PREPARE

TEMPLATE

RUN

SEQUENCE

ANALYZE

DATA

lon Proton™ System

Sample to results in a single day

lon Xpress'" Plus Fragment Library Kits

lon Chef '" System•

or ion OneTouch'" 2 System

lon Proton'"

Sequencer Proton'" Torrent Server

Life Technologies™ I 7



Ion PI™ Chip for Whole Exomes/Transcriptomes; Ion PII™ Chip for Whole Genomes

Ion PI™ Chip

165 M wells

Up to 10 Gb Exome

60-80M Filtered Reads

Up to 200bp reads

2-4 hours

Ion PII™ Chip

660 M wells

Up to 20X human genome

240-320M Filtered Reads

Up to 200bp reads

Runs in Hours



Se

qu

en

ce

Ou

tpu

t p

er

Ru

n

Unprecedented Scalability 10,000-Fold From Ion 314™ to Ion PIII™ Chip

100G

Small Genome

Small to Large Gene Panels

Transcriptome

Exome

Human Genome

Ion PIII™

10G

Ion PII™

Ion PI™

1G

Ion 318™

100M

Ion 316™

10M Ion 314

from 1.2 Million Sensors …………………to 1.2 Billion Sensors

Simple and Complete Workflows

Life Technologies™ |

10

Ion PGM™ System 10

Ion Proton™ System


hnologoes lrwllrogen A.!:lpt edBto,pte m G1bc0 Mott'NI.OrPiobH" Novu T qM:III"" Amboon !onTOO"rent


lon Semiconductor Sequencing:

Enabling a Breadth of Applications

SEQU£NCING S1ALL SETS OF G£NE EXPRtSSION WHOLE HUMAN HUMAN APPLICAnONS GENOMES GENES CHIP SEQ TRANSCRIPTOME EXOMES GENOMES

SEQUENCING OHIPS El PI Pll

Life Technologies™ I 11



Ion Reporter Software Workflow

A secure, hosted informatics infrastructure for routine assays

Automated informatics User interpretation

Reads Mapped Reads

Variants Annotated Variants

Confident

Variants

Relevant

Variants Interpretive

Report



All products mentioned in this presentation are for research use only and not for use in diagnostic procedures.

All data shown in this presentation was generated by Life Technologies, except where indicated

© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies

Corporation and/or its affiliate(s) or their respective owners.

Fast isogenic mapping-by-

sequencing of EMS-induced

mutant bulks

Franziska Turck Group Max Planck

Institute for Plant Breeding Research

14

Presentation Outline

Introduction to technical and biological

problem

Isogenic mapping approach

Fast mapping using deep candidate

resequencing (dCARE) with IonPGMTM

15

Mutations in LHP1/TFL2 alter plant

development

SD/LD

lhp1

lhp1 ft

WT lhp1

AG

SEP3

AP3

AP1

CD

Hinge CSD

binds to H3K27me3 16

LHP1 represses target genes as part

of Polycomb Repressive Complex 1

bmi1a; bmi1b

BMI1c

RING1B

BMI1a

BMI1b

?

lhp1; ring1a;

Bratzel et al. (2010) Chen et al. (2010), Olmo et al. (2010), Adrian et al. (2009), Xu and Shen

RING1A

LHP1

BMI1c

1b

lhp

ring1a;

1b

(2008), Barrero et al.

(2007)

17

Common problems in genetic screens

lethality of strong enhancers

11D at

16°C +

30LDs

Col-0 lhp1

18-8-2

44LDs

Col-0 lhp1

18-8-2

second allele of lhp1 in Ws can only be used

as pollen donor

variance between accessions greater than

effect of weak enhancers and suppressors

18

EMS

Inverse mapping principle

EMS

Back-crossing to parental mutants solves all problems due to

variation between accessions 19

Phenotype of sup3

Col-0 sup3 lhp1

36 LDs Col-0 sup3 lhp1

Col-0 sup3 lhp1

• Plant size is

increased

• Rosette leaves,

Cauline leaves

and siliques are

bigger than in

lhp1 mutants

• Flowers look

the same in

screening

conditions

Col-0 sup3 lhp1

36d, 12h

light,

16°C/day,

14°C/ night 20

Experimental strategy:

Back-cross suppressor 2x to original lhp1 line

Pick 270 individuals with suppressor phenotype

in the F2 of BC2

Prepare DNA from bulked suppressor mutants

Sequence 1 lane on Solexa GAIl or any other

high-throughput sequencing device

Sequence also original ems mutant lhp1 (bulk

of 58)

Analyse with SHORE map

21

Homozygous

SNPs

All SNPs

No. reliable SNPs lhp1

vs Col-0 (all / EMS)

1603 / 273 8497 / 1836

No. reliable SNPs Sup3

vs lhp1 (all / EMS)

6 / 3 2125 / 455

Solexa Sequencing and Data Analysis

Cologne Center for Genomics

Geo Velikkakam, IMPRS student

Group Schneeberger MPIPZ

Read Statistics SNP Statistics

Ref_lhp1 sup3

No. raw reads ~ 84 Million ~ 84 Million

No. aligned reads 79.68 Million 78.06 Million

Genomic coverage ~ 49 X ~ 41 X

SHOREmap 22

hromosome 1 Chromosome 2 Chromoso

Parameters:

10 reads and

>10 (values

hromosome 4 Chromosome 5

frequency

frequency

freque

ncy

Frequency distribution

C me 3

at least

a score

0-40)

C

Chromosome 3 23

2-3 candidates

Chromosome 3

Putative causal

21455 Kb 21456 Kb 22622 Kb 22623 Kb 23376 Kb 23377 Kb

mutations AT3G57940

* AT3G63270

DNA sequence TCTCTTCTCCTGAAGGTCGCAAGGGAGTTAT TCTCTTCTCCTGAAGATCGCAAGGGAGTTAT

CTCTTCTCCTGAAGATCGCAAGGGAGTTAT

CTCTTCTCCTGAAGATCGCAAGGGAGTTAT

TCTTCTCCTGAAGATCGCAAGGGAGTTAT

CTTCTCCTGAAGATCGCAAGGGAGTTAT

TTCTCCTGAAGATCGCAAGGGAGTTAT

TCTCTTCTCCTGAAGATCGCAAGGGAGTTA

TCTCTTCTCCTGAAGATCGCAAGGGAGTT

TCTCTTCTCCTGAAGATCGCAAGGGAGTT

TCTCTTCTCCTGAAGATCGCAAGGGAGT

TCTCTTCTCCTGAAGATCGCAAGGGAG

CGGGTAACTGATCCCTCCAACAACGTATTCTC

CGGGTAACTGATCCCTTCAACAACGTATTCTC

GGGTAACTGATCCCTTCAACAACGTATTCTC



GTAACTGATCCCTTCAACAACGTATTCTC

TAACTGATCCCTTCAACAACGTATTCTC

AACTGATCCCTTCAACAACGTATTCTC

AACTGATCCCTTCAACAACGTATTCTC CGGGTAACTGATCCCTTCAACAACGTATTCT CGGGTAACTGATCCCTTCAACAACGTATTC CGGGTAACTGATCCCTTCAACAACGTATT

Protein change G Q I H S L L L K VA R E L Y K Y L N

GQI HSLLLK I ARELYKYLN

SQGAQIREYVVGGISYPLLP

SQGAQIREYVV E GISYPLLP

25

Mapping strategy the slow way

High resolution

melting PCR

isolate DNA from single

sup3 plants Mapping with 2-3 two markers

26

Fast mapping using deep

Candidate resequening (dCARE)

270 mutants were pooled in initial screen

coverage by whole-genome re-sequencing was 40-fold on average

Distance between candidate loci was at least 2 Megabases, therefore

several recombination events should be present in the pool (average

rate in Arabidopsis is 3 centi-Morgan by Megabase)

Why not sequence the candidates to greater depths to recover a clear

signal from the recombination events?

27

Mu

tati

on

fre

qu

en

cy

Deeper coverage results in higher confidence

prediction of rare recombination events

1.02

1

0.98

0.96

0.94

0.92

0.9

0.88

0.86

0.84

dCARE distinguishes the causal from closely linked mutations

coverage at 40-fold

at 20000-fold

AT3G57940 AT3G61130 AT3G63270

Deep candidate resequencing (dCARE)

Deep sequencing with Ion PGMTM 314K chip

-Relatively small coverage was still sufficient for a limited number of amplicons

-Fast, almost overnight results

-Easy design of library using extended primers

120-250bp

1-50bp

Deep candidate resequencing (dCARE)

Seq-run Pos SNP Cov A C G T N

Cov Freq Cov Freq Cov Freq Cov Freq Cov Freq

AT3G57940 A 50 47 0,940 0 0,000 2 0,040 0 0,000 1 0,020

Illumina

AT3G61130 T 48 0 0,000 2 0,042 0 0,000 44 0,917 2 0,042

AT3G63270 T 41 0 0,000 1 0,024 0 0,000 39 0,951 1 0,024

AT3G57940 A 20111 18966 0,943 0 0 1145 0,057 1 0 0 0

4390 0 0 90 0,02 0 0 4300 0,979 0 0

Here we

have a

winner

AT3G61130 T

dCARE AT3G63270 T 19203 0 0 86 0,005 0 0 19117 0,996 0 0

14-6-1 m uta tion [W 141S top] 15-4-3 1 m uta tion [G273E]

sup3♀ sup3♀ sup2♀ sup2♀ sup1♀ sup1♀

X X X X X X sup2♂ sup1♂ sup3♂ sup1♂ sup3♂ sup2♂

2nd Allele of sup3

sup2 mu [W141Stop] sup3 mu [G273E]

238

At3g63270 cDNA 1260 bp

237

Found a GA change in sup2

coding for a stop codon

sup3

like

F2

F1 30

Nu

mb

er

of

leaves

Complementation of sup3 and sup2 but

not sup1 with 35S:AT3G63270:HA

Flowering time of T1 and non-transformed plants in LDs

Rosette leaves T1 Rosette leaves Cauline leaves T1 Cauline leaves 25

20

15

10

5

0

Col-0 lhp1 sup1 sup2 sup3 Col-0 lhp1 sup1 sup2 sup3

Plants

31

SUP3 enodes a domesticated Harbinger-

like transposase

Protein blast on NCBI

Chose first 100 proteins

from all species

Chose A.t. proteins with

E-value < 0.05 (0.0003)

Bootstrapped neighbor joining

tree with 10.000 replicates

SUP3 does not cluster with

“real” transposons

32

Differential gene expression in lhp1

and sup3

Genes up-regulated compared to Col-0

FDR > 0,05 / logFC > 2

AGL79, MYB18, MYB43,

HSP70, AP1, AGL44, MYB45

lhp1 (286) sup3 (82)

FUL, AGO3, HB21, SPL5, MYB85,

AGL19, STM

252 34 48

SEP1, SEP2, SEP3, AP3, AG, PI,

PRR3, FLC 33

ALP1 – a Trithorax Group protein?

Suppresso

r

ALP1

H3K4me3 adapted from T. Zografou

ALP1 LHP1

PRC1

components

LHP1 ALP1

Target Gene

34

Perspectives

3 suppressors (all sequenced and mapped)

16 enhancers (3 sequenced and mapped, 2

worked with dCARE, one worked down to

two candidates)

One back-cross was sufficient, 100 mutants

in the pool was also o.k.

Barcoding and pooling of Illumina High-seq

re-sequencing with lower coverage was o.k.

Safe cost in whole genome sequencing and do

dCARE for more candidates 35

Turck Group:

Theo Zografou

Korbinian

Collaborations:

Justin Goodrich

Liangyu Liu

Tingting Ning

Petra Tänzler

Yue Zhou

former members

Sara Farrona

Jessika Adrian

Jian Zhang

Benjamin Hartwig

Julia Engelhorn

Julia Reimer

Schneeberger Geo Velikkakam

MPIPZ

Shih Chieh-Liang (Edinburgh University)

Thank you!

37

Visit the Life Technologies website for more information.

http://www.lifetechnologies.com/us/en/home.html?CID=slideshare-plant-sequencing-webinar-12112012

Enhancers on plates

15-9-1 / 2 plants in bulk

16-2-1_1 / 1 plant in bulk

Date post:	25-Dec-2014
Category:	Technology
Upload:	life-technologies
View:	1,150 times
Download:	2 times

Semiconductor Sequencing Applications for Plant Sciences

Technology