Development of a high-throughput sequencing infrastructure for the analysis of complex genomes

With the importance of rice as a sophisticated genetic model plant, CCW as a member of IRGSP was established in 1999 and funded to sequence the short arms of chromosomes 10 and 3. This dissertation research was initiated as a precursor to the establishment of the CCW Rice Genome project and discussed over-all strategies and procedures used for completing rice chromosome 10 sequencing using chromosome 10p0.0∼10p4.4. To apply the clone-by-clone shotgun sequencing strategy, I developed a sequence-ready map of rice ( Oryza sativa ssp. japonica cv. Nipponbare) chromosome 10p0.0∼10p4.4 containing 515 unique bands (CB units) containing a total of 432 BAC clones, and 35 anchored markers for large scale sequencing of the region. A total 21 BACs including 17 CUGI BACs and 4 Monsanto BACs were selected and sequenced using the complete sequencing-ready map by STC strategy. The total sequenced size for rice chromosome 10p0.0∼10p4.4 was 2,331,000 bp and the sequenced overlaps for the region were 736,354 bp of the total sequence with average overlap of 36,817 bp. Using two annotation methods from the years 2001 and 2003, the 2,331,000 bp was characterized to the sequence level and the results from both of the pipelines were compared. Domain-based paralogous families were identified using HMM search against Pfam, and repetitive sequence was analyzed using public database (grass repeat database).;By integrating in silico fingerprints of finished and phase II draft BAC sequences into the current FPC map, the CUGI rice FPC map (Chen et al. 2002) was revised. A new AGI rice physical map, representing 93% of the genome, with 84 physical gaps, has been generated and was released to the public (http://www.genome.arizona.edu/fpc/rice/) on September 3rd, 2003.;To establish an efficient high throughput strategy for the project, two methods were developed: a simplified high-throughput shotgun library construction procedure and high-throughput template DNA preparation method which provides speed, cost effectiveness, and high quality.