Procedure Normalisation DMD MLPA 89files

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					 Genetic Testing in Europe - FP6 Network of Excellence

                 Network for test development harmonization, validation
                 and standardization of services



               Evaluation of the performance of the existing tools
                             for MLPA data analysis
Laboratory :
Address:
Person in charge of the validation :
e-mail:
Phone number:


Objective
The objective of the second part of MLPA validation is to evaluate the performance of the existing Excel sheets
and software for MLPA data analysis.
In this respect, a set of pre-existing MLPA files of capillary electrophoresis peak profiles will be analysed by
the MLPA working group using their own excel sheets or software.


Location of the DMD Files

A selection of MLPA files after screening for copy number changes in the DMD gene were kindly provided by
Leiden University Medical Center, the Netherlands (Dr. Bakker).
89 files contain MLPA information for the DMD gene after screening by the P034 and/orP035 MLPA kits from
MRC-Holland.

The DMD MLPA files, organized in 6 folders can be downloaded from the following link:
(http://www.eurogentest.org/cocoon/egtorg/web/info/public/unit1/MLPAvalidation.xhtml)-


Details about the P034 and P035 are available in the document “SALSA MLPA kit P034/P035 DMD/ becker”
provided by MRC-holland at the following URL address :
(http://www.mrc-holland.com/pages/p034___p035pag.html)



Procedure for the analysis of the DMD Files


   DMD files folder is divided into 6 subfolders:

   DMD-P034-1
   DMD-P035-2
   DMD-P034-3
   DMD-P035-4
   DMD-P034-5
   DMD-P035-6

Each of the 6 above folders contains a number of samples for which MLPA reactions were run together. All the 6
MLPA runs include :

   -      A “no template controls called “blanco”
   -      2 negative controls (D1.03.02235DMD and D1.03.02609DMD, in red in the excel sheet created to report
          the data; see below)
   -      2 positive controls (D1.03.01831DMD and D1.04.04280DMD, in green in the excel sheet)
   -      A number of various tested samples with or without copy number changes.

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 Genetic Testing in Europe - FP6 Network of Excellence

                   Network for test development harmonization, validation
                   and standardization of services




Samples within subfolders ending with 1,3 and 5 were analysed using the P034 kit and samples within
subfolders ending with 2, 4 and 6 using the P035 kit.
Respectively, the subfolders 1, 2, 3, 4, 5 and 6 contain 17, 12, 14, 11, 20, 15 files.

Analyse independently the 6 groups of samples located in the 6 different subfolders and normalise the data
using the data from the 2 negative controls mentioned above.

Please note that we added in subfolders 5 and 6, 4 samples that gave unclear MLPA traces. Two of them named
QA.05.00019DMD and QA.05.00020DMD were confirmed with an extra method.



Report of the results


An excel sheet called ‘Report_results_MLPA_normalisation_ 89DMD traces’ is sent to all participants via email
or can also be found at the following URL address:
(http://www.eurogentest.org/cocoon/egtorg/web/info/public/unit1/MLPAvalidation.xhtml)

Please report in the corresponding excel tables :
    - The sex (M/F)
    - When a sample contains copy number changes, which DMD exons are duplicated or deleted
    - When no copy number changes were identified, “NO copy number changes”
    - When no conclusions could have been made, “Non-conclusive”


Normalisation of the data

The normalisation of the MLPA data are based on :

    -    Peak areas
    -    Peak heights
    -    Others

Please, provide a detailed description of the model you use for the normalisation of the MLPA data




We would be grateful if you could return the data to Florence Le Calvez via email
                                                st
(florence.lecalvez@uz.kuleuven.ac.be), by the 1 of March 2006 and even before if you could.


Florence Le Calvez,
Laboratory for Molecular Diagnosis
Center For Human Genetics
Herestraat 49
B-3000 Leuven
Belgium
Tel: + 32 (0) 16 34 79 51
Fax: + 32 (0) 16 34 79 49
Email: florence.lecalvez@uz.kuleuven.ac.be




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