Salehi-Ashtiani Kourosh, Yang Xinping, Derti Adnan, Tian Weidong, Hao Tong, Lin Chenwei, Makowski Kathryn, Shen Lei, Murray Ryan R, Szeto David, Tusneem Nadeem, Smith Douglas R, Cusick Michael E, Hill David E, Roth Frederick P, Vidal Marc
Center for Cancer Systems Biology, Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.
Nat Methods. 2008 Jul;5(7):597-600. doi: 10.1038/nmeth.1224. Epub 2008 Jun 15.
Describing the 'ORFeome' of an organism, including all major isoforms, is essential for a system-level understanding of any species; however, conventional cloning and sequencing approaches are prohibitively costly and labor-intensive. We describe a potentially genome-wide methodology for efficiently capturing new coding isoforms using reverse transcriptase (RT)-PCR recombinational cloning, 'deep-well' pooling and a next-generation sequencing platform. This ORFeome discovery pipeline will be applicable to any eukaryotic species with a sequenced genome.
描述生物体的“开放阅读框组”(ORFeome),包括所有主要的异构体,对于从系统层面理解任何物种而言至关重要;然而,传统的克隆和测序方法成本过高且 labor-intensive。我们描述了一种潜在的全基因组方法,该方法利用逆转录酶(RT)-PCR 重组克隆、“深孔”合并和下一代测序平台来有效捕获新的编码异构体。这种 ORFeome 发现流程将适用于任何具有已测序基因组的真核物种。