Medical Genetics Institute, Shaare Zedek Medical Center, 91031, Jerusalem, Israel.
Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel.
Mol Genet Genomics. 2021 May;296(3):653-663. doi: 10.1007/s00438-021-01772-3. Epub 2021 Mar 11.
Next generation sequencing tests are used routinely as first-choice tests in the clinic. However, systematic performance comparing the results of exome sequencing as a single test replacing Sanger sequencing of targeted gene(s) is still lacking. Performance comparison data are critically important for clinical case management. In this study, we compared Sanger-sequencing results of 258 genes to those obtained from next generation sequencing (NGS) using two exome-sequencing enrichment kits: Agilent-SureSelectQXT and Illumina-Nextera. Sequencing was performed on leukocytes and buccal-derived DNA from a single individual, and all 258 genes were sequenced a total of 11 times (using different sequencing methods and DNA sources). Sanger sequencing was completed for all exons, including flanking ± 8 bp regions. For the 258 genes, NGS mean coverage was > 20 × for > 98 and > 91% of the regions targeted by SureSelect and Nextera, respectively. Overall, 449 variants were identified in at least one experiment, and 407/449 (90.6%) were detected by all. Of the 42 discordant variants, 23 were determined as true calls, summing-up to a truth set of 430 variants. Sensitivity of true-variant detection was 99% for Sanger sequencing and 97-100% for the NGS experiments. Mean false-positive rates were 3.7E-6 for Sanger sequencing, 2.5E-6 for SureSelect-NGS and 5.2E-6 for Nextera-NGS. Our findings suggest a high overall concordance between Sanger sequencing and NGS performances. Both methods demonstrated false-positive and false-negative calls. High clinical suspicion for a specific diagnosis should, therefore, override negative results of either Sanger sequencing or NGS.
下一代测序测试通常作为临床首选测试。然而，作为一种替代靶向基因(s) 桑格测序的单一测试，外显子组测序的系统性能比较仍然缺乏。性能比较数据对于临床病例管理至关重要。在这项研究中，我们比较了使用两种外显子组测序富集试剂盒：Agilent-SureSelectQXT 和 Illumina-Nextera，对单个个体的白细胞和口腔衍生 DNA 进行的桑格测序和下一代测序 (NGS) 的结果。所有 258 个基因总共测序了 11 次（使用不同的测序方法和 DNA 来源）。桑格测序完成了所有外显子，包括侧翼 ± 8 bp 区域。对于 258 个基因，NGS 的平均覆盖率分别超过了 SureSelect 和 Nextera 靶向区域的 98%和 91%以上。总体而言，在至少一个实验中鉴定了 449 个变体，并且所有变体都检测到了 407/449（90.6%）。在 42 个不一致的变体中，有 23 个被确定为真实变体，总共有 430 个变体的真实变体集合。桑格测序检测真实变体的灵敏度为 99%，NGS 实验的灵敏度为 97-100%。桑格测序的平均假阳性率为 3.7E-6，SureSelect-NGS 为 2.5E-6，Nextera-NGS 为 5.2E-6。我们的研究结果表明，桑格测序和 NGS 性能之间具有高度的总体一致性。两种方法都表现出假阳性和假阴性的结果。因此，对于特定诊断的高临床怀疑应该超过桑格测序或 NGS 的阴性结果。
