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Laboratory Management |
1 Institute for Clinical Chemistry and 2
the Department for Statistical Analysis, University Hospital Mannheim of the University of Heidelberg, Mannheim, Germany.
3 German Society for Clinical Chemistry and Laboratory Medicine (DGKL) Reference-Institute for Bioanalytics, Bonn, Germany.
4 National Genetics Reference Laboratory (Manchester), St Mary’s Hospital, Manchester, United Kingdom.
5 Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.
aAddress correspondence to this author at: Institute for Clinical Chemistry, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany. Fax 49-621-383-3819; e-mail michael.neumaier{at}ikc.ma.uni-heidelberg.de.
Background: DNA sequencing is a key technique in molecular diagnostics, but to date no comprehensive methodologic external quality assessment (EQA) programs have been instituted. Between 2003 and 2005, the European Union funded, as specific support actions, the EQUAL initiative to develop methodologic EQA schemes for genotyping (EQUALqual), quantitative PCR (EQUALquant), and sequencing (EQUALseq). Here we report on the results of the EQUALseq program.
Methods: The participating laboratories received a 4-sample set comprising 2 DNA plasmids, a PCR product, and a finished sequencing reaction to be analyzed. Data and information from detailed questionnaires were uploaded online and evaluated by use of a scoring system for technical skills and proficiency of data interpretation.
Results: Sixty laboratories from 21 European countries registered, and 43 participants (72%) returned data and samples. Capillary electrophoresis was the predominant platform (n = 39; 91%). The median contiguous correct sequence stretch was 527 nucleotides with considerable variation in quality of both primary data and data evaluation. The association between laboratory performance and the number of sequencing assays/year was statistically significant (P <0.05). Interestingly, more than 30% of participants neither added comments to their data nor made efforts to identify the gene sequences or mutational positions.
Conclusions: Considerable variations exist even in a highly standardized methodology such as DNA sequencing. Methodologic EQAs are appropriate tools to uncover strengths and weaknesses in both technique and proficiency, and our results emphasize the need for mandatory EQAs. The results of EQUALseq should help improve the overall quality of molecular genetics findings obtained by DNA sequencing.
The following articles in journals at HighWire Press have cited this article:
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  J. Marki-Zay, C. L. Klein, D. Gancberg, H. G. Schimmel, and L. Dux European External Quality Control Study on the Competence of Laboratories to Recognize Rare Sequence Variants Resulting in Unusual Genotyping Results Clin. Chem., April 1, 2009; 55(4): 739 - 747. [Abstract] [Full Text] [PDF]
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 B. Afshar, N. K. Fry, W. Bellamy, A. P. Underwood, T. G. Harrison, and and Members of the European Working Group for Legi External Quality Assessment of a DNA Sequence-Based Scheme for Epidemiological Typing of Legionella pneumophila by an International Network of Laboratories J. Clin. Microbiol., October 1, 2007; 45(10): 3251 - 3256. [Abstract] [Full Text] [PDF]
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C. Orlando, P. Verderio, R. Maatman, J. Danneberg, S. Ramsden, M. Neumaier, D. Taruscio, V. Falbo, R. Jansen, C. Casini-Raggi, et al. EQUAL-qual: A European Program for External Quality Assessment of Genomic DNA Extraction and PCR Amplification Clin. Chem., July 1, 2007; 53(7): 1349 - 1357. [Abstract] [Full Text] [PDF]
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A. Dorn-Beineke, P. Ahmad-Nejad, U. Pfeiffer, S. Ramsden, M. Pazzagli, and M. Neumaier Improvement of Technical and Analytical Performance in DNA Sequencing by External Quality Assessment-Based Molecular Training Clin. Chem., November 1, 2006; 52(11): 2072 - 2078. [Abstract] [Full Text] [PDF]
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S. C. Ramsden, S. Daly, W.-J. Geilenkeuser, G. Duncan, F. Hermitte, E. Marubini, M. Neumaier, C. Orlando, V. Palicka, A. Paradiso, et al. EQUAL-quant: An International External Quality Assessment Scheme for Real-Time PCR Clin. Chem., August 1, 2006; 52(8): 1584 - 1591. [Abstract] [Full Text] [PDF]
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E. Bakker Is the DNA sequence the gold standard in genetic testing? Quality of molecular genetic tests assessed. Clin. Chem., April 1, 2006; 52(4): 557 - 558. [Full Text] [PDF]
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