Semiconductor Sequencing Applications for Plant Sciences

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

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



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lon Semiconductor Sequencing:
Enabling a Breadth of Applications
SEQU£NC
ING S1ALL SETS OF G£NE EXPRtSSION WHOLE HUMAN HUMAN
APPLICAnONS GENOMES GENES CHIP SEQ TRANSCRIPTOME EXOMES GENOMES

SEQUENCNG
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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

AG
SEP3
AP3
WT lhp1 AP1

CD Hinge CSD

binds to H3K27me3 16

LHP1 represses target genes as part
of Polycomb Repressive Complex 1

bmi1a; bmi1b
BMI1c

BMI1b
Bratzel et al. (2010) Chen
BMI1a et al. (2010), Olmo et al.
RING1B
(2010), Adrian et al.
? lhp1; ring1a; (2009), Xu and Shen
RING1A (2008), Barrero et al.
1b
(2007)

LHP1 BMI1c ring1a;
1b
lhp 17

Common problems in genetic screens

 lethality of strong enhancers
11D at 44LDs
16°C +
30LDs
Col-0 lhp1
Col-0 lhp1 18-8-2
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

Inverse mapping principle
EMS EMS

Back-crossing to parental mutants solves all problems due to
variation between accessions 19

Phenotype of sup3
Col-0 sup3 lhp1
36 LDs • Plant size is
increased
• Rosette leaves,
Cauline leaves
and siliques are
bigger than in
lhp1 mutants
• Flowers look
the same in
screening
Col-0 sup3 lhp1 Col-0 sup3 lhp1 conditions

36d, 12h
light,
16°C/day,
20
Col-0 sup3 lhp1 14°C/ night

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

Solexa Sequencing and Data Analysis

Geo Velikkakam,
IMPRS student
Cologne Center for Genomics
Group Schneeberger MPIPZ

Read Statistics SNP Statistics
Ref_lhp1 sup3 Homozygous All SNPs
SNPs
No. raw reads ~ 84 Million ~ 84 Million
No. reliable SNPs lhp1 1603 / 273 8497 / 1836
vs Col-0 (all / EMS)
No. aligned reads 79.68 Million 78.06 Million
No. reliable SNPs Sup3 6/3 2125 / 455
Genomic coverage ~ 49 X ~ 41 X vs lhp1 (all / EMS)

SHOREmap 22

Frequency distribution
frequency

frequency

C hromosome 1 Chromosome 2 Chromosome 3

Parameters: at least
10 reads and a score
frequency

>10 (values 0-40)

Chromosome 4 Chromosome 5
23
Chromosome 3

2-3 candidates
Chromosome 3

21455 Kb 21456 Kb 22622 Kb 22623 Kb 23376 Kb 23377 Kb
Putative causal
AT3G57940 AT3G63270
mutations
*

DNA sequence TCTCTTCTCCTGAAGGTCGCAAGGGAGTTAT CGGGTAACTGATCCCTCCAACAACGTATTCTC
TCTCTTCTCCTGAAGATCGCAAGGGAGTTAT CGGGTAACTGATCCCTTCAACAACGTATTCTC
CTCTTCTCCTGAAGATCGCAAGGGAGTTAT GGGTAACTGATCCCTTCAACAACGTATTCTC
CTCTTCTCCTGAAGATCGCAAGGGAGTTAT GGGTAACTGATCCCTTCAACAACGTATTCTC
TCTTCTCCTGAAGATCGCAAGGGAGTTAT GGGTAACTGATCCCTTCAACAACGTATTCTC
CTTCTCCTGAAGATCGCAAGGGAGTTAT GTAACTGATCCCTTCAACAACGTATTCTC
TTCTCCTGAAGATCGCAAGGGAGTTAT TAACTGATCCCTTCAACAACGTATTCTC
TCTCTTCTCCTGAAGATCGCAAGGGAGTTA AACTGATCCCTTCAACAACGTATTCTC
TCTCTTCTCCTGAAGATCGCAAGGGAGTT AACTGATCCCTTCAACAACGTATTCTC
TCTCTTCTCCTGAAGATCGCAAGGGAGTT CGGGTAACTGATCCCTTCAACAACGTATTCT
TCTCTTCTCCTGAAGATCGCAAGGGAGT CGGGTAACTGATCCCTTCAACAACGTATTC
TCTCTTCTCCTGAAGATCGCAAGGGAG CGGGTAACTGATCCCTTCAACAACGTATT

Protein change G Q I H S L L L K VA R E L Y K Y L N SQGAQIREYVVGGISYPLLP
GQI HSLLLK I ARELYKYLN SQGAQIREYVV E GISYPLLP

Mapping strategy the slow way

High resolution
melting PCR

isolate DNA from single
Mapping with 2-3 two markers
sup3 plants

25

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?

26

Deeper coverage results in higher confidence
prediction of rare recombination events

dCARE distinguishes the causal from closely linked
mutations
1.02
1
0.98
Mutation frequency

0.96
0.94
coverage at 40-fold
0.92
at 20000-fold
0.9
0.88
0.86
0.84
AT3G57940 AT3G61130 AT3G63270

27

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

AT3G61130 T 48 0 0,000 2 0,042 0 0,000 44 0,917 2 0,042
Here we
Illumina AT3G63270 T 41 0 0,000 1 0,024 0 0,000 39 0,951 1 0,024
have a
AT3G57940 A 20111 18966 0,943 0 0 1145 0,057 1 0 0 0 winner
4390 0 0 90 0,02 0 0 4300 0,979 0 0
AT3G61130 T

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

2nd Allele of sup3

sup2 mu [W141Stop] 1 msup3[G273E]
14-6-1 m uta tion [W 141S top] 15-4-3 uta tion mu [G273E]

238

At3g63270 cDNA 237
1260 bp

Found a GA change in sup2
coding for a stop codon

sup3
like

F2

sup3♀ sup3♀ sup2♀ sup2♀ sup1♀ sup1♀
X X X X X X 30
F1 sup2♂ sup1♂ sup3♂ sup1♂ sup3♂ sup2♂

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
Number of leaves

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, FUL, AGO3, HB21, SPL5, MYB85,
HSP70, AP1, AGL44, MYB45 lhp1 (286) sup3 (82) 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

LHP1 ALP1

Target Gene
PRC1
components 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: Collaborations:
Korbinian Justin Goodrich
Theo Zografou
Schneeberger Shih Chieh-Liang
Liangyu Liu Geo Velikkakam (Edinburgh University)
Tingting Ning MPIPZ

Petra Tänzler
Yue Zhou
former members
Sara Farrona
Jessika Adrian
Jian Zhang
Benjamin Hartwig
Julia Engelhorn
Julia Reimer

Thank you!

Visit the Life Technologies website for more information.

37

Enhancers on plates

15-9-1 / 2 plants in bulk

16-2-1_1 / 1 plant in bulk

Semiconductor Sequencing Applications for Plant Sciences

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