Replication timing data analysis in Neuroblastoma


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Preferential Occurrence of Chromosome Breakpoints within Early Replicating Regions in Neuroblastoma.
Isabelle Janoueix-Lerosey1, Philippe Hupé2,4, Zofia Maciorowski3, Philippe La Rosa4, Gudrun Schleiermacher1, Gaelle Pierron5, Stéphane Liva4, Emmanuel Barillot4 and Olivier Delattre1

1 INSERM U509, Laboratoire de Pathologie Moléculaire des Cancers, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France
2 UMR 144 CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France
3 Service de Cytométrie, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France
4 Service Bioinformatique, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France
5 Unité de Genétique Somatique, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France





This replication timing data analysis was based on the GLAD algorithm (Hupé et al, 2004) and the VAMP software (La Rosa et al, submitted) developed by the Bioinformatics group from Institut Curie.



Abstract
Data description
Data visualization withing VAMP
Guidelines to visualize data:
1 - Replication Timing data
2 - Comparison between Sanger's data and Curie's data
3 - Array CGH data
4 - Comparison between breakpoints position and replication timing
Raw Data




Abstract

Neuroblastoma (NB) is a frequent paediatric extra cranial solid tumour whose genetic hallmark is the occurrence of unbalanced translocations. Previous analysis of NB cell lines using chromosomal CGH and 24-colour karyotyping suggested that the majority of the breakpoints corresponding to these unbalanced translocations mapped to early replicating chromosome bands. In order to better evaluate the relationship between the position of the breakpoints corresponding to unbalanced translocations and replication timing in NB cell lines, we have now used genome wide arrays containing 3400 PAC/BAC clones spaced at approximately 1 Mb intervals: (1) to further refine the mapping of breakpoints in 28 NB cell lines by a classical array-CGH method; (2) to assess replication timing of sequences during the S phase of the cell cycle in 7 NB cell lines, using the method recently described by K. Woodfine et al (2004).

Data description

Data corresponding to these experiments are visualized using VAMP (Visualization and Analysis of CGH array, transcriptome and other Molecular Profiles) and organized into 4 folders:


The BREAKPOINTS folder contains the position of the 142 breakpoints associated to colour transition and gain or loss of genetic material by cCGH that have been further mapped by array-CGH. Breakpoint positions were determined using the DAGLAD algorithm (Hupé et al, manuscript in preparation), a recently modified version of GLAD (Hupé et al, 2004).


The CELL-LINES-CGH folder contains the array-CGH profiles for the 28 NB cell lines.

The CELL-LINES-RT folder contains the replication timing profiles for the 7 cell lines : for each cell line, 2 profiles were obtained since replication timing was evaluated after a sort of the total S phase or a sort of the first quarter of the S phase (S1). This folder also contains the Average-Total S phase and Average-S1 profiles obtained after calculating an average replicating ratio for the 7 samples, in total S or S1 fractions, respectively.

The SANGER-RT folder contains the replication timing profile drawn from the data obtained by K. Woodfine et al (2004) on a human lymphoblastoid cell line of normal karyotype after a total S phase sort.

Each folder includes 2 subfolders, “arrays” and “chromosomes” that enable the visualization of the whole genome profiles or the visualization of only a selected chromosome, respectively.



Data visualization within VAMP

You have to launch the VAMP interface clicking the button below:


then use File->Import to load data


vamp

Warning: configure your java virtual machine according the following instructions. Your browser must allow java to be used.


Guidelines to visualize data

Some guidelines are given below to help the user visualizing the data within VAMP interface:
1 - Replication Timing data
2 - Comparison between Sanger's data and Curie's data
3 - Array CGH data
4 - Comparison between breakpoints position and replication timing

1 - Replication Timing Data

Close the previous window and open a new window

View -> New View -> Double View -> Point View

Import the averaged replication timing data

File -> Import -> In Top Panel -> CELL-LINES-RT -> Arrays->  average-TotalS
View -> Current View -> Top Panel -> Gained/Lost Color Codes

Import the other replication timing array data

File -> Import -> In Bottom Panel -> CELL-LINES-RT -> Arrays 
(and select the array you want to visualize then click on Import data)
View -> Current View -> Bottom Panel -> Gained/Lost Color Codes
Red = early replicated
Green = Late replicated


2 - Comparison between Sanger's data and Curie's data

Close the previous window and open a new window

View -> New View -> Simple View -> Point View
File -> Import -> SANGER-RT -> Arrays -> SANGER-RT
File -> Import -> CELL-LINES-RT -> Arrays -> average-TotalS
View -> Current View -> GNL Color Codes
View -> Current View -> Show Smoothing Line
Scale the data in Y axis

3 - Array CGH data

Import all the array CGH data

File -> Import -> CELL-LINES-CGH -> Arrays
Select All the arrays and then click on Import data

Increase the scale in Y axis and X axis

Add the results of the analysis by the GLAD algorithm

View -> Current View -> Show Smoothing Line
View -> Current View -> Show Breakpoints
View -> Current View -> Gained/Lost Color Codes
Green = Loss
Yellow = Normal
Red = Gain
Blue = Amplicon

Select and copy all the array CGH data

Edit -> Select All and Copy

Paste the data in karyotype view

View -> New View -> Simple View -> Karyotype Classic View
Edit -> Paste
View -> Current View -> Gained/Lost Color Codes
View -> Current View -> Show Smoothing Line
Scale the data in Y axis

N.B. The default behaviour is to plot the data in LogRatio. If you want to switch to Ratio then:
Edit -> Select All
Tools -> CGH -> Change CGH Ratio L to M

4 - Comparison between breakpoints position and replication timing

Open a new window

View -> New View -> Double View -> Point View

Import the averaged replication timing data

File -> Import -> In Bottom Panel -> CELL-LINES-RT -> Arrays -> average-TotalS
View -> Current View -> Bottom Panel -> Gained/Lost Color Codes


Import the breakpoins data

File -> Import -> In Top Panel -> BREAKPOINTS -> Arrays -> Breakpoints
Click on Y-range and set Max to 2 and Apply


Hightlight the region early replicated

Tools -> In Bottom Panel -> CGH -> Minimal Alteration
Select "widens regions"
unselect "Amplicon" and "Lost"
Click on OK



Raw Data


Raw data (gpr files) for the 28 cell line array CGH profiles can de downloaded here.


The breakpoints detection has been performed using the GLAD algorithm
Vamp Project : http://bioinfo.curie.fr/projects/vamp
Contact : vamp@curie.fr Home Page : http://bioinfo.curie.fr

To download the java plugin click here