Ahmad-Nejad Parviz, Dorn-Beineke Alexandra, Pfeiffer Ulrike, Brade Joachim, Geilenkeuser Wolf-Jochen, Ramsden Simon, Pazzagli Mario, Neumaier Michael
Institute for Clinical Chemistry and the Department for Statistical Analysis, University Hospital Mannheim of the University of Heidelberg, Mannheim, Germany.
Clin Chem. 2006 Apr;52(4):716-27. doi: 10.1373/clinchem.2005.061572. Epub 2006 Jan 26.
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.
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.
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.
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.
DNA测序是分子诊断中的一项关键技术，但迄今为止尚未建立全面的方法学外部质量评估（EQA）计划。2003年至2005年期间，欧盟作为特定支持行动资助了EQUAL计划，以开发用于基因分型（EQUALqual）、定量PCR（EQUALquant）和测序（EQUALseq）的方法学EQA方案。在此，我们报告EQUALseq计划的结果。
参与实验室收到一组包含2个DNA质粒、1个PCR产物和1个已完成测序反应的4个样本进行分析。详细问卷中的数据和信息在线上传，并使用技术技能和数据解释熟练度评分系统进行评估。
来自21个欧洲国家的60个实验室注册，43名参与者（72%）返回了数据和样本。毛细管电泳是主要平台（n = 39；91%）。连续正确序列延伸的中位数为527个核苷酸，原始数据和数据评估的质量均存在相当大的差异。实验室表现与每年测序检测次数之间的关联具有统计学意义（P <0.05）。有趣的是，超过30%的参与者既没有对其数据添加注释，也没有努力识别基因序列或突变位置。
即使在DNA测序这种高度标准化的方法中也存在相当大的差异。方法学EQA是发现技术和熟练度方面优缺点的合适工具，我们的结果强调了强制性EQA的必要性。EQUALseq的结果应有助于提高通过DNA测序获得的分子遗传学研究结果的整体质量。
