Department of Human Genetics, Saarland University, Building 60, 66421, Homburg/Saar, Germany.
Clinical Bioinformatics, Saarland University, Building E2.1, 66123, Saarbrücken, Germany.
J Mol Med (Berl). 2019 Aug;97(8):1099-1111. doi: 10.1007/s00109-019-01792-y. Epub 2019 May 27.
Gene amplification is an evolutionarily well-conserved and highly efficient mechanism to increase the amount of specific proteins. In humans, gene amplification is a hallmark of cancer and has recently been found during stem cell differentiation. Amplifications in stem cells are restricted to specific tissue areas and time windows, rendering their detection difficult. Here, we report on the performance of deep WGS sequencing (average 82-fold depth of coverage) on the BGISEQ with nanoball technology to detect amplifications in human mesenchymal and neural stem cells. As reference technology, we applied array-based comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), and qPCR. Using different in silico strategies for amplification detection, we analyzed the potential of WGS for amplification detection. Our results provide evidence that WGS accurately identifies changes of the copy number profiles in human stem cell differentiation. However, the identified changes are not in all cases consistent between WGS and aCGH. The results between WGS and the validation by qPCR were concordant in 83.3% of all tested 36 cases. In sum, both genome-wide techniques, aCGH and WGS, have unique advantages and specific challenges, calling for locus-specific confirmation by the low-throughput approaches qPCR or FISH. KEY MESSAGES: WGS allows for the identification of dynamic copy number changes in human stem cells. Less stringent threshold setting is crucial for detection of copy number increase. Broad knowledge of dynamic copy number is pivotal to estimate stem cell capabilities.
基因扩增是一种进化上保守且高效的机制，可增加特定蛋白质的数量。在人类中，基因扩增是癌症的一个标志，最近在干细胞分化过程中也发现了这种现象。干细胞中的扩增仅限于特定的组织区域和时间窗口，这使得它们的检测变得困难。在这里，我们报告了使用基于纳米球技术的深度 WGS 测序（平均覆盖深度 82 倍）在 BGISEQ 上检测人类间充质和神经干细胞中扩增的性能。作为参考技术，我们应用了基于阵列的比较基因组杂交（aCGH）、荧光原位杂交（FISH）和 qPCR。我们使用不同的扩增检测计算策略来分析 WGS 进行扩增检测的潜力。我们的结果表明，WGS 可以准确识别人类干细胞分化过程中拷贝数谱的变化。然而，在 WGS 和 aCGH 之间，并非所有情况下都能观察到一致的变化。在所有测试的 36 个病例中，WGS 和 qPCR 验证的结果有 83.3%是一致的。总之，全基因组技术 aCGH 和 WGS 都有独特的优势和特定的挑战，需要通过低通量方法 qPCR 或 FISH 进行特定基因座的确认。关键信息：WGS 允许识别人类干细胞中动态的拷贝数变化。更严格的阈值设置对于检测拷贝数增加至关重要。广泛了解动态拷贝数对于评估干细胞能力至关重要。
