Chung Jongsuk, Son Dae-Soon, Jeon Hyo-Jeong, Kim Kyoung-Mee, Park Gahee, Ryu Gyu Ha, Park Woong-Yang, Park Donghyun
Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology, Samsung Electronics Co. Ltd., Seoul 06351, Korea.
Samsung Genome Institute, Samsung Medical Center, Seoul 06351, Korea.
Sci Rep. 2016 May 25;6:26732. doi: 10.1038/srep26732.
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.
靶向捕获大规模平行测序在临床环境中的应用日益广泛，且随着成本持续下降，这项技术在医疗保健领域的使用可能会成为常规操作。然而，在满足质量要求方面，组织样本量有限常常是一项挑战。为了提供关于靶向测序所需最低输入DNA量的实用指南，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，我们使用不同量的输入DNA，比较了市售的文库构建试剂盒，并选择了安捷伦的SureSelect-XT试剂盒和卡帕生物系统公司的Hyper Prep试剂盒，作为与使用安捷伦SureSelect定制捕获进行靶向深度测序最兼容的试剂盒。然后，我们优化了Hyper Prep试剂盒的接头连接条件，以提高文库构建效率，并采用多重杂交选择来降低测序成本。在本研究中，我们系统地评估了根据输入DNA量（范围为6.25至200 ng）优化后的方案的性能，根据特定应用所需的覆盖深度提出了最低输入DNA量。
