Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Uppsala, Sweden.
Nucleic Acids Res. 2011 Jan;39(2):e8. doi: 10.1093/nar/gkq1005. Epub 2010 Nov 8.
Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R(2) = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.
靶向基因组富集是利用新型 DNA 测序仪器高通量的强大工具。我们在此提出了一种基于 Selector 技术的靶向区域多重扩增的简单且可扩展的方案。该更新版本提高了覆盖率，并且与新一代测序 (NGS) 文库构建程序更兼容，适用于 NGS 平台的鸟枪法测序。为了展示该技术的性能，我们对源自细胞系和肿瘤活检的样本中经常涉及癌症的 28 个基因的 501 个外显子进行了富集和测序。对新鲜冷冻和福尔马林固定石蜡包埋活检的 DNA 进行了分析，实现了靶向区域 94%的特异性和 98%的覆盖率。重复之间的重现性很高（R(2) = 0.98），并且可以轻松检测拷贝数变异。该方案可以在<24 小时内完成，不需要任何专用仪器。
