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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
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∆ Q
Sensing Layer
Sensor Plate
∆ V
Bulk Drain Source
Silicon Substrate To column
receiver
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lon Semiconductor Sequencing:
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SEQU£NCING S1ALL SETS OF G£NE EXPRtSSION WHOLE HUMAN HUMAN APPLICAnONS GENOMES GENES CHIP SEQ TRANSCRIPTOME EXOMES GENOMES
SEQUENCING OHIPS El PI Pll
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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
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
GGGTAACTGATCCCTTCAACAACGTATTCTC
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
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Enhancers on plates
15-9-1 / 2 plants in bulk
16-2-1_1 / 1 plant in bulk