Finish of Clone-By-Clone Sequencing of Wheat 6B Chromosome with Next-Generation Sequencer

The International Wheat Genome Sequencing Consortium (IWGSC) is making efforts to decipher the map-based genomic sequence of bread wheat (Triticum aestivum L. cv. Chinese Spring). As a member of IWGSC, we have constructed a BAC physical map with the Whole Genome Profiling (WGP) method using the wheat chromosome 6B-specific BAC library, and then about 8,544 minimal tiling path (MTP) clones were selected. Based on the IWGSC policy, we have established a high throughput system by using the next-generation sequencer (NGS) to sequence wheat chromosome 6B with a clone-by-clone method. Here we report the application of this sequencing system to the MTP BAC clones of wheat 6B. Throughput enhancements are essential for efficient sequencing with a large number of BAC clones. Therefore, BAC DNAs were prepared with 96 deep-well box, and they were tagged with 48 or 96 multiple indexes (MIDs). Tagged BAC DNAs were sequenced with Roche GS FLX and the obtained sequences were assembled with GS assembler. We obtained about 70Gb sequence data with GS FLX from 8,428 of 8,544 applied MTP BAC clones (x52.5 /BAC clone). This sequencing system with tagged library and GS FLX is quite useful for sequencing with BAC clones on a larger scale. Based on the sequences obtained here, we will make a high quality reference sequence for wheat chromosome 6B. This work was supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan. (KGS1001, KGS1003 and NGB1003)