Clark M D, Hennig S, Herwig R, Clifton S W, Marra M A, Lehrach H, Johnson S L
Max-Planck-Institut für Molekulare Genetik, 14195 Berlin, Germany.
Genome Res. 2001 Sep;11(9):1594-602. doi: 10.1101/gr.186901.
The zebrafish is a powerful system for understanding the vertebrate genome, allowing the combination of genetic, molecular, and embryological analysis. Expressed sequence tags (ESTs) provide a rapid means of identifying an organism's genes for further analysis, but any EST project is limited by the availability of suitable libraries. Such cDNA libraries must be of high quality and provide a high rate of gene discovery. However, commonly used normalization and subtraction procedures tend to select for shorter, truncated, and internally primed inserts, seriously affecting library quality. An alternative procedure is to use oligonucleotide fingerprinting (OFP) to precluster clones before EST sequencing, thereby reducing the re-sequencing of common transcripts. Here, we describe the use of OFP to normalize and subtract 75,000 clones from two cDNA libraries, to a minimal set of 25,102 clones. We generated 25,788 ESTs (11,380 3' and 14,408 5') from over 16,000 of these clones. Clustering of 10,654 high-quality 3' ESTs from this set identified 7232 clusters (likely genes), corresponding to a 68% gene diversity rate, comparable to what has been reported for the best normalized human cDNA libraries, and indicating that the complete set of 25,102 clones contains as many as 17,000 genes. Yet, the library quality remains high. The complete set of 25,102 clones is available for researchers as glycerol stocks, filters sets, and as individual EST clones. These resources have been used for radiation hybrid, genetic, and physical mapping of the zebrafish genome, as well as positional cloning and candidate gene identification, molecular marker, and microarray development.
斑马鱼是用于理解脊椎动物基因组的强大系统，它能将遗传分析、分子分析和胚胎学分析结合起来。表达序列标签（EST）为鉴定生物体基因以供进一步分析提供了一种快速方法，但任何EST项目都受到合适文库可用性的限制。此类cDNA文库必须质量高且能提供高基因发现率。然而，常用的标准化和消减程序往往会选择较短、截短和内部引发的插入片段，严重影响文库质量。另一种方法是在EST测序之前使用寡核苷酸指纹图谱（OFP）对克隆进行预聚类，从而减少常见转录本的重复测序。在此，我们描述了使用OFP对来自两个cDNA文库的75,000个克隆进行标准化和消减，得到最少25,102个克隆的集合。我们从这些克隆中的16,000多个克隆中生成了25,788个EST（11,380个3'端和14,408个5'端）。对该集合中10,654个高质量3'端EST进行聚类，鉴定出7232个簇（可能的基因），对应68%的基因多样性率，与报道的最佳标准化人类cDNA文库相当，这表明25,102个克隆的完整集合包含多达17,000个基因。然而，文库质量仍然很高。25,102个克隆的完整集合以甘油菌液、滤膜组和单个EST克隆的形式提供给研究人员。这些资源已用于斑马鱼基因组的辐射杂种、遗传和物理图谱绘制，以及定位克隆和候选基因鉴定、分子标记和微阵列开发。
