Construction of medaka BAC library

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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.