The minimal amount of starting DNA for Agilent's hybrid capture-based targeted massively parallel sequencing.
The minimal amount of starting DNA for Agilent's hybrid capture-based targeted massively parallel sequencing.
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DOI：
10.1038/srep26732
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发表时间：
2016-05-25
影响因子：
4.6
通讯作者：
Park D
中科院分区：
文献类型：
--
作者：
Chung J;Son DS;Jeon HJ;Kim KM;Park G;Ryu GH;Park WY;Park D
靶向捕获大规模平行测序越来越多地用于临床环境，随着成本的持续下降，这种技术的使用可能成为医疗保健的常规。然而，在满足质量要求方面，有限数量的组织常常是一个挑战。为了给靶向测序的最小输入DNA量提供实用的指导，我们根据输入DNA量对靶向测序的性能进行了优化和评估。首先，使用不同数量的输入DNA，我们比较了市售的文库构建试剂盒，并选择了Agilent的SureSelect- xt和KAPA Biosystems的Hyper Prep试剂盒，作为使用Agilent的SureSelect自定义捕获的靶向深度测序最兼容的试剂盒。然后，我们优化了Hyper Prep试剂盒的适配器连接条件，以提高文库构建效率，并适应多路杂交选择，以降低测序成本。在本研究中，我们根据输入DNA的数量系统地评估了优化方案的性能，范围从6.25到200 ng，根据特定应用所需的覆盖深度提出了最小输入DNA量。
Targeted capture massively parallel sequencing is increasingly being used in clinical settings, and as costs continue to decline, use of this technology may become routine in health care. However, a limited amount of tissue has often been a challenge in meeting quality requirements. To offer a practical guideline for the minimum amount of input DNA for targeted sequencing, we optimized and evaluated the performance of targeted sequencing depending on the input DNA amount. First, using various amounts of input DNA, we compared commercially available library construction kits and selected Agilent’s SureSelect-XT and KAPA Biosystems’ Hyper Prep kits as the kits most compatible with targeted deep sequencing using Agilent’s SureSelect custom capture. Then, we optimized the adapter ligation conditions of the Hyper Prep kit to improve library construction efficiency and adapted multiplexed hybrid selection to reduce the cost of sequencing. In this study, we systematically evaluated the performance of the optimized protocol depending on the amount of input DNA, ranging from 6.25 to 200 ng, suggesting the minimal input DNA amounts based on coverage depths required for specific applications.