Plant, Animal & Microbe Genomes X Conference

January 12-16, 2002
Town & Country Convention Center
San Diego, CA

Poster: Genome Sequencing & ESTs
           

AN AUTOMATED SHOTGUN TEMPLATE PREPARATION PIPELINE FOR RICE GENOME SEQUENCING

¹ Rice Genome Research Program, National Institute of Agrobiological Sciences / Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan

The International Rice Genome Sequencing Project (IRGSP) has announced in February 2001 a new policy for completing the rice genome sequence in the shortest possible time. To accomplish this goal, the Rice Genome Research Program (RGP) has modified the sequencing strategy (http://rgp.dna.affrc.go.jp/genomicdata/seqstrategy/newstrategy.html) to accelerate the release of phase 2 and phase 3 sequence data. As a result, about 116 Mbp of genome sequence has been submitted to DDBJ as of October 2001. Currently, we have aligned more than 1400 PAC (P1-derived artificial chromosome) / BAC (bacterial artificial chromosome) clones along chromosomes 1, 2, 6, 7 and 8 for shotgun sequencing. In order to accelerate template preparation, we developed a semi-automated system for extraction of plasmid DNA by alkaline lysis and purification with glass-fiber filter based mainly on Biomek 2000. Additionally, we developed a new and improved operating system using Biomek FX. This new system is more efficient and can almost double the output in the template preparation step of sequencing. For each PAC / BAC clone we usually construct 2 kb and 5 kb shotgun libraries each consisting of 960 subclones. Using both systems, we can prepare plasmid DNAs at a constant rate of 90 PAC / BAC clones, or 180,000 subclones per month. This work is supported by the MAFF Rice Genome Project Grant GS-1201.