An oligonucleotide fingerprint normalized and expressed sequence tag characterized zebrafish cDNA library
An oligonucleotide fingerprint normalized and expressed sequence tag characterized zebrafish cDNA library
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DOI：
10.1101/gr.186901
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发表时间：
2001-09-01
期刊：
影响因子：
7
通讯作者：
Johnson, SL
中科院分区：
文献类型：
--
作者：
Clark, MD;Hennig, S;Johnson, SL
斑马鱼是了解脊椎动物基因组的强大系统，允许遗传学，分子学和胚胎学分析相结合。表达序列标签（EST）为进一步分析提供了一种快速识别生物基因的方法，但任何EST项目都受到合适文库可用性的限制。这样的cDNA文库必须是高质量的并且提供高的基因发现率。然而，通常使用的标准化和减法程序倾向于选择较短的、截短的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（OFP）对克隆进行预聚类，从而减少常见转录本的重新测序。在这里，我们描述了使用OFP从两个cDNA文库中标准化并减去75，000个克隆，得到25，102个克隆的最小集合。我们从16，000多个克隆中产生了25，788个EST（11，380个3 '和14，408个5 '）。从这组中聚类10，654个高质量的3 'EST鉴定出7232个簇（可能的基因），对应于68%的基因多样性率，与已报道的最佳标准化的人类cDNA文库相当，并表明完整的25，102个克隆组包含多达17，000个基因。图书馆的质量仍然很高。完整的25，102个克隆可作为甘油储备、过滤器组和单个EST克隆供研究人员使用。这些资源已被用于辐射杂交，遗传和物理作图，斑马鱼基因组，以及定位克隆和候选基因鉴定，分子标记和微阵列的发展。
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.