Plant & Animal Genome IX Conference

Poster: Sequencing & EST
P01_29.html

AN EFFICIENT SYSTEM OF SHOTGUN TEMPLATE PREPARATION FROM PAC / BAC DNA FOR RICE GENOME SEQUENCING

Rice Genome Research Program, National Institute of Agrobiological Resources / Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan

The Rice Genome Research Program (RGP) has processed more than 300 PAC (P1-derived artificial chromosome) / BAC (bacterial artificial chromosome) clones for the past two years as part of the rice genome sequencing effort in collaboration with the International Rice Genome Sequencing Project (IRGSP). As the first step in the preparation of templates for shotgun sequencing, each PAC / BAC clone was purified using the Qiagen column. Subsequently the purified DNA with less than 1% E. coli DNA contamination was fragmented and subcloned using pUC vector. The resulting shotgun library for each PAC / BAC clone consisted of 1920 clones with insert lengths ranging 1.5-2 kb and 5.5-7 kb. The subclones with longer insert could evenly cover the entire PAC / BAC clone and could be effective as bridges between the sequence contigs. All subclones were cultured in minimal volume in 96-well plates and directly subjected to plasmid extraction step. An automated plasmid extraction system with 96-well plates was recently established which enabled us to prepare plasmid DNA at a rate of 50 PAC / BAC clones per month, providing sufficient quality and quantity of templates for sequencing reaction. In case the sequence of each PAC / BAC did not complement, we also used transposon-primed sequencing method and nested deletion method to supplement the sequence coverage. As we accelerate the rice genome sequence analysis, various procedures are constantly being improved to maintain a stable supply of optimized sequencing templates. This work is supported by the MAFF Rice Genome Project Grant GS-1201.