Oshikawa Mio, Sugai Yoshiko, Usami Ron, Ohtoko Kuniyo, Toyama Shigeru, Kato Seishi
Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for Persons with Disabilities, 4-1 Namiki, Tokorozawa, Saitama 359-8555, Japan.
DNA Res. 2008 Jun 30;15(3):123-36. doi: 10.1093/dnares/dsn010. Epub 2008 May 16.
Recently, we have developed a vector-capping method for constructing a full-length cDNA library. In the present study, we performed in-depth analysis of the vector-capped cDNA library prepared from a single type of cell. As a result of single-pass sequencing analysis of 24,000 clones randomly isolated from the unamplified library, we identified 19,951 full-length cDNA clones whose intactness was confirmed by the presence of an additional G at their 5' end. The full-length cDNA content was >95%. Mapping these sequences to the human genome, we identified 4,513 transcriptional units that include 36 antisense transcripts against known genes. Comparison of the frequencies of abundant clones showed that the expression profiles of different libraries, including the distribution of transcriptional start sites (TSSs), were reproducible. The analysis of long-sized cDNAs showed that this library contained many cDNAs with a long-sized insert up to 11,199 bp of golgin B, including multiple slicing variants for filamin A and filamin B. These results suggest that the size-unbiased full-length cDNA library constructed using the vector-capping method will be an ideal resource for fine expression profiling of transcriptional variants with alternative TSSs and alternative splicing.
最近，我们开发了一种用于构建全长cDNA文库的载体加帽方法。在本研究中，我们对从单一类型细胞制备的载体加帽cDNA文库进行了深入分析。通过对从未扩增文库中随机分离的24000个克隆进行单通道测序分析，我们鉴定出19951个全长cDNA克隆，其完整性通过在其5'端存在额外的G得以证实。全长cDNA含量>95%。将这些序列映射到人类基因组，我们鉴定出4513个转录单元，其中包括针对已知基因的36个反义转录本。对丰富克隆频率的比较表明，不同文库的表达谱，包括转录起始位点（TSS）的分布，是可重复的。对长片段cDNA的分析表明，该文库包含许多插入片段长达11199 bp的高尔基体蛋白B的长片段cDNA，包括细丝蛋白A和细丝蛋白B的多个剪接变体。这些结果表明，使用载体加帽方法构建的大小无偏全长cDNA文库将是用于具有替代TSS和替代剪接的转录变体精细表达谱分析的理想资源。
