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Semi-Automated Unidirectional Sequence Analysis for Mutation Detection in a Clinical Diagnostic Setting

机译：半自动单向序列分析突变检测在临床诊断设置

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摘要

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. Methods: 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. Results: A total of 701 different heterozygous base substitutions were detected by the software with no false negatives (sensitivity >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 >30 and an average PHRED-like score >50 for the defined region of interest, and 98.1% of the 323,650 bases had a PHRED score >40. Conclusions: 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.
机译：过去十年已经看到的改善由于引入序列数据质量毛细管测序和新的测序化学反应。自动化的突变检测发展。半自动的单向序列分析杂合的检测的基础使用突变测量员替换软件包。杂合的基础在29基因替换自动化和视觉检验进行比较。杂合的基地没有检测到的例子在双向分析一个方向也寻求通过一个统一的全国性调查王国(英国)遗传学实验室。在一个连续的质量评估50个病人来说,ABCC8的39个外显子基因测序在一个方向。结果:共有701个不同的杂合的基地替换被检测到该软件没有假阴性(> 99.57%)的敏感性。错过了一个四个例子的杂合的基地在双向分析方向报道。分析设置,和其他两个基地低质量分数。检查,有97.2%质量分数> 30和一个平均PHRED-like得分> 50的定义感兴趣的区域,和323650年的98.1%基地有phr得分> 40岁。没有发现证据支持要求双向测序。优质的单向数据序列灵敏度高,适用于杂合子突变扫描在临床诊断实验室。确定最低质量参数半自动的分析。

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