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Development of high-‐density NimbleGen microarray chip for genotyping of a radia9on-‐hybrid panel of wheat
chromosome 7B
Ta9ana Belova
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no 2
Aim of the study
Develop a CGH NimbleGen high-‐density microarray chip for genotyping RH-‐popula=on and construc=on of a high density RH-‐map of wheat chromosome 7B
Use a subset of dele=on lines to construct 7B chromosome bin map
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Radiation hybrid panel
3
Langdon 7D(7B) Langdon Chr 1-6: AABB Chr 1-7: AABB Chr 7: AADD
1200 RH1 panel for chromosome 7B Chr 1-6: AABB Chr 7 : AABD
300RH1 lines selected with retention frequency 10-90%
Irradiate seeds at 25,35,45 krad
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Design of probes for NimbleGen microarray
4
Mask sequences for repeats (TREP10 and repeats iden=fied in Choulet et al.2011)
-‐ Blast against 7D and 7A -‐ No homopolymers of >=8 -‐ Tm between 66 and 86
Extract 50bp sequences
Run sequences through ISBP finder,
Remove low confidence ISBPs
Extract 24bp from both sides of junc=on
Scaffolds/con=gs assembled from PE and MP data
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Identifying 7B specific probes
5
3*720K CGH chip
12*270K CGH chip
7B dele=on bins and reference (Langdon)
LDN
LDN 7D(7B)
49,500 7B specific probes (5,465 ISBP and 44,035 random genomic fragments)
18,000 control probes
7D(7B)
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Normalization of Microarray Data
- To correct for systematic differences between samples on the same slide, or between slides, which do not represent true biological variation between samples but arise due to:
different labelling efficiencies of the dyes
different amounts of Cy3- and Cy5-labeled DNA
differences in scanning
print-tip, spatial, effects, etc.
6
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Two-step normalization of CGH-arrays
7
Spatial intensity normalization
- =
Print-tip Lowess normalization
- Produce MA plot
M=log(cy3)-log(cy5)
A=(log(cy3)+log(cy5))/2
- Apply a Lowess regression to the data
- Calculate the normalized M value by
M normalized=M original-M predicted by Loess
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
How does Lowess work?
8
• Lowess performs a large number of local regressions in overlapping windows
• Each regression is then then combined to form a smooth curve
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Selecting 7B specific and control probes
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0 20000 40000 60000
−3−2
−10
12
3
Index
log2
(LD
N)−
log(
7B−)
conserved (58132) ctrl (10000) ISBP (7197)
9
log(
LDN
)-lo
g (7
B-)
RGF control ISBP
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Identification of bin probes
10
-1.0 -0.5 0.0 0.5 1.0
0.10.2
0.30.4
0.50.6
0.70.8
x
y
-1.0 -0.5 0.0 0.5 1.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
x
y
0
ratio (del/ref)
Probes absent in deletion line
Probes present in deletion line and reference
NO
RWEG
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NIVERSITY O
F LIFE SCIENCES
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Averaging over probe replicates vs scaffolds
11
On probes
On scaffolds (>3 probes)
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Identification of present/absent probes in deletion lines
12
Absent probes
Mclust in R
NO
RWEG
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NIVERSITY O
F LIFE SCIENCES
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Deletion bin mapping
13
Absent probe
Probe is filtered out from dataset
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Deletion bin map of 7B
14
7BL-‐0.14
7BL-‐0.14-‐0.33
7BL-‐0.33-‐0.45
7BL-‐0.45-‐0.63
7BL-‐0.63-‐0.86
7BL-‐0.86
probes contigs scaffolds
2413 1211 339
2914 1465 472
1565 750 233
1842 894 293
365 196 68
1718 859 304
NO
RWEG
IAN U
NIVERSITY O
F LIFE SCIENCES
www.umb.no
Summary
Developed CGH microarray chip will be used for genotyping radiation-hybrid panel
We have bin mapped 5357 contigs (1709 scaffolds with >3 probes) for 7BL
10817 probes were bin mapped for 7BL among which 1396 ISBPs and 9421 RGF
15
NO
RWEG
IAN U
NIVERSITY O
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Collaborators
16
Norwegian University of Life Sciences (UMB) Odd-Arne Olsen Simen R. Sandve Bujie Zhan Lars Gronvold
University of Minnesota Nathan Springer
North Dakota State University Shahryar Kianian Ajay Kumar
TGAC
Jonathan Wright
University of Queensland, Australia
David Edwards
International Wheat Genome Consortium
NO
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