Construction of medaka BAC library

1. 从近交系HNI菌株中构建了一套高质量的Medaka基因组BAC文库。大约96,000个平均插入长度为160 kb的克隆覆盖了medaka基因组的20倍。这些克隆体分别储存在384孔板中的250个孔板中。这些无性系也被发现在尼龙膜上进行集落杂交筛选。我们建立了一种高效获取bac端序列的新方法，使用Qiagen R.E.A.L. Prep 96制备bac - dna，并通过ABI3700/3730毛细管测序仪直接测序。我们完成了11,904个Hd-rR BAC克隆、8,160个HNI BAC克隆和1,152个Cab BAC克隆的末端测序。总共42,432个末端序列数据作为GSS （Genome survey sequence）存储在数据库中。建立了Hd-rR BAC文库的两步四维（4D-）PCR筛选系统。第一次筛选是针对超级池，每个超级池由1536个BAC克隆组成。然后，第二次筛选是针对第一次筛选确定的阳性超级池。每个超级池的单个克隆被定位为4维，因此我们可以通过28秒池的PCR反应从1,536个克隆中识别单个克隆。我们准备了20个超级池，因此总共有30,720个克隆可访问，对应于Medaka基因组的8倍。一套筛选可以在一天内完成。我们与MGI的几个研究小组合作，开始了medaka LG22染色体的全测序（约20 Mb）。我们已经用BAC contigs覆盖了11Mb的基因组区域，完成了7Mb的染色体测序。迄今为止，我们向几个研究小组提供了许多BAC克隆、筛选材料和BAC末端序列数据。BAC文库对medaka性别决定基因等基因的定位克隆、基因结构分析以及BAC全基因组序列图和连锁图的构建做出了贡献。

1. We have constructed a set of high-quality Medaka genome BAC library from the inbred HNI strain. About 96,000 clones with average insert size of 160 kb cover 20 times the medaka genome. These clones were individually stocked in 250 of 384-well plates. These clones were also spotted on nylon membranes for colony hybridization screening.2. We have established a novel procedure to efficiently obtain BAC-end sequences, in which the BAC-DNAs are prepared with 96-well format using Qiagen R.E.A.L. Prep 96 and are sequenced directly by ABI3700/3730 capillary sequencers. We have finished the end-sequencing of 11, 904 Hd-rR BAC clones, 8,160 HNI BAC clones, and 1,152 Cab BAC clones. The total 42,432 end sequence data were deposited in our database as GSS (Genome survey sequence).3. We also established the two step four dimensional (4D-)PCR screening system for the Hd-rR BAC library. First screening is against superpools each consisting of 1,536 BAC clones. Then, second screening is against the positive superpools identified by the first screening. Individual clones of each superpool are addressed 4-dimensionally, therefore we can identify single clone from the l,536 clones by PCR reactions for 28 second pools. We have prepared twenty superpools, therefore total 30,720 clones are accessible which correspond to 8 times of Medaka genome. A set of screening can be finished within a day.4. We started entire sequencing of the medaka LG22 chromosome (about 20 Mb) in collaboration with several research groups in the MGI. We have covered 11Mb of genomic region with BAC contigs and have finished sequencing 7Mb of the chromosome.5. To date, we provided many BAC clones, materials for screenings, and BAC-end sequence data to several research groups. It is noted that the BAC libraries have contributed positional cloning of several genes including medaka sex determining gene and analysis of gene structure as well as constructions of genome-wide BAC contig map and linkage map.