Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants.
Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants.
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DOI：
10.1373/clinchem.2015.249623
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发表时间：
2016-04
影响因子：
9.3
通讯作者：
Biesecker LG
中科院分区：
文献类型：
--
作者：
Beck TF;Mullikin JC;NISC Comparative Sequencing Program;Biesecker LG
下一代测序（NGS）数据用于临床护理和临床研究。使用NGS鉴定的DNA序列变体通常作为临床或研究方案的一部分返回给患者/参与者。目前的护理标准是使用桑格测序来验证NGS变体，这是昂贵且耗时的。我们使用来自ClinSeq®项目的数据对NGS变体的基于Sanger的验证进行了大规模的系统评价。我们首先使用了来自5名参与者的19个基因的NGS数据，将其与相同样本的高通量桑格测序结果进行比较，发现234个NGS变体之间没有差异。然后，我们将来自684名参与者的5个基因中的NGS变体与桑格测序数据进行了比较。在超过5,800个NGS衍生的变体中，有19个没有经过桑格数据的验证。使用新设计的测序引物，桑格测序确认了17种NGS变体，其余两种变体的外显子组测序质量评分较低。总体而言，我们使用桑格测序测量了NGS变体的99.965%的验证率，这高于许多不需要正交验证的现有医学测试。单轮桑格测序更有可能错误地反驳来自NGS的真阳性变体，而不是正确地鉴定来自NGS的假阳性变体。使用桑格测序验证NGS衍生变体的效用有限，并且最佳实践标准不应包括NGS变体的常规正交桑格验证。
Next-generation sequencing (NGS) data are used for both clinical care and clinical research. DNA sequence variants identified using NGS are often returned to patients/participants as part of clinical or research protocols. The current standard of care is to validate NGS variants using Sanger sequencing, which is costly and time-consuming. We performed a large-scale, systematic evaluation of Sanger-based validation of NGS variants using data from the ClinSeq® project. We first used NGS data from 19 genes in five participants, comparing them to high-throughput Sanger sequencing results on the same samples, and found no discrepancies among 234 NGS variants. We then compared NGS variants in five genes from 684 participants against data from Sanger sequencing. Of over 5,800 NGS-derived variants, 19 were not validated by Sanger data. Using newly-designed sequencing primers, Sanger sequencing confirmed 17 of the NGS variants, and the remaining two variants had low quality scores from exome sequencing. Overall, we measured a validation rate of 99.965% for NGS variants using Sanger sequencing, which was higher than many existing medical tests that do not necessitate orthogonal validation. A single round of Sanger sequencing is more likely to incorrectly refute a true positive variant from NGS than to correctly identify a false positive variant from NGS. Validation of NGS-derived variants using Sanger sequencing has limited utility, and best practice standards should not include routine orthogonal Sanger validation of NGS variants.