http://www.clinchem.org/cgi/content/abstract/52/4/716

(Clinical Chemistry. 2006;52:716-727.)

Methodologic European External Quality Assurance for DNA Sequencing: The EQUALseq Program

Parviz Ahmad-Nejad^1,1, Alexandra Dorn-Beineke^1,1, Ulrike Pfeiffer¹, Joachim Brade², Wolf-Jochen Geilenkeuser³, Simon Ramsden⁴, Mario Pazzagli⁵ and Michael Neumaier^1,a

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@... .

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