Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory.
Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory.
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DOI：
10.1038/gim.2013.183
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发表时间：
2014-07
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桑格测序目前被认为是临床分子诊断测试的金标准方法。然而，下一代测序（NGS）已经成为鉴定基因组、外显子组或基因组内遗传变异的更有效手段。我们试图评估我们的临床基因组学实验室中NGS变体鉴定的准确性，目的是为基于Sanger的验证性检测建立质量评分阈值。分析了来自144个连续临床外显子组测序病例（94个独特变体）和来自可比研究样本的另外一组16个变体的报告结果的确认数据。110个SNV中103个SNV的质量评分≥Q500，其中103个（100%）经桑格测序证实。在质量评分<Q500的其余7个变体中，6个通过桑格测序确认（85%）。对于单核苷酸变异，我们预测我们将能够减少我们的桑格确认工作量的70- 80%。这证明了一个原则，即只要实施足够的验证和质量控制措施，就可以减少桑格确认的数量，从而减轻希望利用NGS技术的临床实验室的大量劳动力和成本负担。然而，在我们的实验室中，低质量单核苷酸变体和所有插入缺失（插入或缺失小于10 bp）的桑格确认仍然是必要的。
Sanger sequencing is currently considered the gold standard methodology for clinical molecular diagnostic testing. However, next generation sequencing (NGS) has already emerged as a much more efficient means to identify genetic variants within gene panels, the exome, or the genome. We sought to assess the accuracy of NGS variant identification in our clinical genomics laboratory with the goal of establishing a quality score threshold for confirmatory Sanger-based testing. Confirmation data for reported results from 144 sequential clinical exome sequencing cases (94 unique variants) and an additional set of 16 variants from comparable research samples were analyzed. 103 of 110 total SNVs analyzed had a quality score ≥Q500, 103 (100%) of which were confirmed by Sanger sequencing. Of the remaining 7 variants with quality scores <Q500, 6 were confirmed by Sanger sequencing (85%). For single nucleotide variants, we predict we will be able to reduce our Sanger confirmation workload going forward by 70–80%. This serves as a proof of principle that as long as sufficient validation and quality control measures are implemented, the volume of Sanger confirmation can be reduced, alleviating a significant amount of the labor and cost burden on clinical laboratories wishing to utilize NGS technology. However, Sanger confirmation of low quality single nucleotide variants and all indels (insertions or deletions less than 10 bp) remains necessary at this time in our laboratory.
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