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Development of highdensity NimbleGen microarray chip for genotyping of a radia9onhybrid panel of wheat chromosome 7B Ta9ana Belova

Developmentof*high/density* NimbleGen* microarray*chip*for

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Page 1: Developmentof*high/density* NimbleGen* microarray*chip*for

Development  of  high-­‐density  NimbleGen  microarray  chip  for  genotyping  of  a  radia9on-­‐hybrid  panel  of  wheat  

chromosome  7B  

Ta9ana  Belova  

Page 2: Developmentof*high/density* NimbleGen* microarray*chip*for

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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  

Page 3: Developmentof*high/density* NimbleGen* microarray*chip*for

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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

Page 4: Developmentof*high/density* NimbleGen* microarray*chip*for

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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    

Page 5: Developmentof*high/density* NimbleGen* microarray*chip*for

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F LIFE SCIENCES

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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)

Page 6: Developmentof*high/density* NimbleGen* microarray*chip*for

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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

Page 7: Developmentof*high/density* NimbleGen* microarray*chip*for

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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

Page 8: Developmentof*high/density* NimbleGen* microarray*chip*for

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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    

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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

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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

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Averaging over probe replicates vs scaffolds

11

On probes

On scaffolds (>3 probes)

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Identification of present/absent probes in deletion lines

12

Absent probes

Mclust in R

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Deletion bin mapping

13

Absent probe

Probe is filtered out from dataset

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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

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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

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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

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