Samorodnitsky Eric, Datta Jharna, Jewell Benjamin M, Hagopian Raffi, Miya Jharna, Wing Michele R, Damodaran Senthilkumar, Lippus Juliana M, Reeser Julie W, Bhatt Darshna, Timmers Cynthia D, Roychowdhury Sameek
Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio.
Division of Medical Oncology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
J Mol Diagn. 2015 Jan;17(1):64-75. doi: 10.1016/j.jmoldx.2014.09.009.
Targeted, capture-based DNA sequencing is a cost-effective method to focus sequencing on a coding region or other customized region of the genome. There are multiple targeted sequencing methods available, but none has been systematically investigated and compared. We evaluated four commercially available custom-targeted DNA technologies for next-generation sequencing with respect to on-target sequencing, uniformity, and ability to detect single-nucleotide variations (SNVs) and copy number variations. The technologies that used sonication for DNA fragmentation displayed impressive uniformity of capture, whereas the others had shorter preparation times, but sacrificed uniformity. One of those technologies, which uses transposase for DNA fragmentation, has a drawback requiring sample pooling, and the last one, which uses restriction enzymes, has a limitation depending on restriction enzyme digest sites. Although all technologies displayed some level of concordance for calling SNVs, the technologies that require restriction enzymes or transposase missed several SNVs largely because of the lack of coverage. All technologies performed well for copy number variation calling when compared to single-nucleotide polymorphism arrays. These results enable laboratories to compare these methods to make informed decisions for their intended applications.
靶向捕获DNA测序是一种经济高效的方法，可将测序重点聚焦于基因组的编码区域或其他定制区域。目前有多种靶向测序方法，但尚未对其进行系统的研究和比较。我们评估了四种用于新一代测序的市售定制靶向DNA技术，比较了它们在靶向测序、均一性以及检测单核苷酸变异（SNV）和拷贝数变异方面的能力。使用超声破碎DNA的技术在捕获均一性方面表现出色，而其他技术的制备时间较短，但均一性较差。其中一种使用转座酶进行DNA片段化的技术存在需要样本合并的缺点，最后一种使用限制性内切酶的技术则受限于限制性内切酶的酶切位点。尽管所有技术在检测SNV时都表现出一定程度的一致性，但需要限制性内切酶或转座酶的技术由于覆盖不足而遗漏了一些SNV。与单核苷酸多态性阵列相比，所有技术在检测拷贝数变异方面都表现良好。这些结果使实验室能够比较这些方法，以便为其预期应用做出明智的决策。
