Ellard Sian, Shields Beverley, Tysoe Carolyn, Treacy Rebecca, Yau Shu, Mattocks Christopher, Wallace Andrew
Department of Molecular Genetics, Royal Devon & Exeter NHS Foundation Trust, Exeter, United Kingdom.
Genet Test Mol Biomarkers. 2009 Jun;13(3):381-6. doi: 10.1089/gtmb.2008.0096.
The past 10 years have seen an improvement in sequence data quality due to the introduction of capillary sequencers and new sequencing chemistries. In parallel, new software programs for automated mutation detection have been developed. We evaluated the sensitivity of semiautomated unidirectional sequence analysis for the detection of heterozygous base substitutions using the Mutation Surveyor software package.
Detection rates for heterozygous base substitutions in 29 genes by automated and visual inspection were compared. Examples of heterozygous bases not detected in one direction during bidirectional analysis were also sought through a national survey of United Kingdom (UK) genetics laboratories. Sequence quality was assessed in a consecutive cohort of 50 patients for whom the 39 exons of the ABCC8 gene had been sequenced in one direction.
A total of 701 different heterozygous base substitutions were detected by the software with no false negatives (sensitivity >or=99.57%). Four examples of heterozygous bases missed in one direction during bidirectional analysis were reported. Two were detected using unidirectional analysis settings, and the other two bases had low-quality scores. Of the 1950 amplicons examined, 97.2% had a quality score >or=30 and an average PHRED-like score >or=50 for the defined region of interest, and 98.1% of the 323,650 bases had a PHRED score >40.
We found no evidence to support a requirement for bidirectional sequencing. Semiautomated analysis of good quality unidirectional sequence data has high sensitivity and is suitable for heterozygote mutation scanning in clinical diagnostic laboratories. Further work is required to determine minimum quality parameters for semiautomated analysis.
由于毛细管测序仪的引入和新的测序化学方法，过去十年序列数据质量有所提高。与此同时，用于自动突变检测的新软件程序也已开发出来。我们使用Mutation Surveyor软件包评估了半自动单向序列分析检测杂合碱基替换的敏感性。
比较了通过自动检测和目视检查对29个基因中杂合碱基替换的检测率。还通过对英国遗传学实验室的全国性调查，寻找双向分析中在一个方向未检测到的杂合碱基的例子。对50例连续患者进行队列研究，评估ABCC8基因39个外显子单向测序的序列质量。
该软件共检测到701种不同的杂合碱基替换，无假阴性（敏感性≥99.57%）。报告了双向分析中在一个方向遗漏的4个杂合碱基例子。其中2个使用单向分析设置检测到，另外2个碱基质量分数较低。在检测的1950个扩增子中，97.2%在定义的感兴趣区域质量分数≥30，平均类PHRED分数≥50，在323,650个碱基中98.1%的PHRED分数>40。
我们没有发现支持双向测序必要性的证据。高质量单向序列数据的半自动分析具有高敏感性，适用于临床诊断实验室的杂合子突变扫描。需要进一步开展工作来确定半自动分析的最低质量参数。
