A minimal tiling path cosmid library for functional analysis of the Pseudomonas aeruginosa PAO1 genome
A minimal tiling path cosmid library for functional analysis of the Pseudomonas aeruginosa PAO1 genome
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DOI：
10.1089/mcg.2000.5.189
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发表时间：
2000-01-01
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影响因子：
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通讯作者：
Mattick, John S.
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文献类型：
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作者：
Huang, Bixing;Whitchurch, Cynthia B.;Mattick, John S.
铜绿假单胞菌是一种重要的病原菌和环境细菌，其中研究最广泛的菌株是PAO 1。使用PAO 1参考粘粒库和最近完成的PAO 1基因组序列，我们已经绘制了一个最小的平铺路径，使用两步策略的基因组。首先，我们对一组超过500个随机和先前映射的克隆的两端进行测序以创建骨架。其次，我们对第二组粘粒克隆进行末端测序，这些克隆被鉴定为位于较大的缺口内，使用参考文库过滤器与针对每个缺口中心序列设计的探针进行杂交。使用程序Domino（http：//www.bit.uq.edu.au/download/）计算最小平铺路径，相邻克隆之间的重叠设置为5 kb（如果可能）以使截短基因的机会最小化。这产生了一个最小的平铺粘粒文库（334个克隆），在57个重叠群中覆盖了93.7%的基因组。该文库将代表大多数铜绿假单胞菌基因组所需的克隆数量减少到一个可行的集合，并给出了每个粘粒的精确位置，使得大多数感兴趣的基因能够定位在克隆上而无需进一步筛选。该文库将被证明是加速铜绿假单胞菌基因组功能分析的有用资源。
Pseudomonas aeruginosa is an important pathogenic and environmental bacterium, with the most widely studied strain being PAO1. Using the PAO1 reference cosmid library and the recently completed PAO1 genome sequence, we have mapped a minimal tiling path across the genome using a two-step strategy. First, we sequenced both ends of a set of over 500 random and previously mapped clones to create a backbone. Second, we end-sequenced a second set of cosmid clones that were identified to lie within the larger gaps using hybridization of the reference library filters with probes designed against sequences at the center of each gap. The minimal tiling path was calculated using the program Domino (http://www.bit.uq.edu.au/download/), with the overlap between adjacent clones set to 5 kb (where possible) to minimize the chance of truncating genes. This yielded a minimal tiling cosmid library (334 clones) covering 93.7% of the genome in 57 contigs. This library has reduced to a workable set the number of clones required to represent the majority of the P. aeruginosa genome and gives the precise location of each cosmid, enabling most genes of interest to be located on clones without further screening. This library should prove a useful resource to accelerate functional analysis of the P. aeruginosa genome.