In this shared instrumentation grant, we are seeking funds to purchase a Roche Genome Sequencer 20 (GS20). This instrument is based on the revolutionary sequencing technology developed at 454 Life Sciences and now distributed through Roche Diagnostics. Described in a Nature article in Sept. 2005 , the GS20 provides approximately two hundred thousand, 100 base pair sequence reads in a single 4.5hr run at a cost of a little over $6,000 in reagents and supplies. That is, one can obtain 20Mbp of sequence, with 2-3 days of sample preparation at a cost that is 100 fold (on a per bp basis) lower than standard electrophoresis based technologies. This technology provides radical new opportunities for high throughput discovery which will be initially applied at the University of Washington in the following areas: 1) Sequencing of clinical isolates and phenotypically interesting strains of previously sequenced bacterial genomes. For many pathogenic bacteria, large collections of clinical isolates with varying pathogenicity exist both at the University of Washington and elsewhere. While a large number of molecular biological techniques exist that one can use to hunt for genomic regions that are correlated with phenotype, all present day methods are time consuming and tedious. Inexpensive sequencing of bacterial genomes will enable a far more efficient identification and characterization of pathogen sequence traits that correlated with clinical outcomes. 2) Characterization of sequence distributions within retroviral populations. Retroviral genomes rapidly mutate within the host in response to both the host defenses and therapy. This sequencing technology will be applied to a gaining a more rapid understanding of viral evolution that will aid in the development of vaccines and treatments. 3) Rapid re-sequencing of selected genomic regions or cDNA's in mammalian genomes. With modest modifications to currently published protocols, this technology will be adapted to high throughput, full length sequencing of pools of cDNA's and to deep re- sequencing of selected genes in a population to identify rare polymorphisms. ? ? ?