In this application, the investigator proposes to determine the entire genomic DNA sequence of the gram positive bacterium Streptococcus pneumoniae. The investigator and her colleagues have worked on various aspects of this organism for many years, and they plan to use their accumulated knowledge and that of the broader S. pneumoniae research community to chose a strain of this organism to sequence (likely the Spanish 23F strain), based on a number of biological and health-related factors. In a close working collaboration with Dr. Ellson Chen's high-throughput sequencing group at A.B.I., the groups will use ordered shotgun sequencing (OSS) to determine the 2.4 Mb genome sequence. Their strategy will be to make a lambda library with inserts approximately 10 kb in length representing the genome of the bacterium, and to sequence the ends of 2300 random clones from the library (representing about 10- fold cloned coverage). A roughly minimum overlapping set of 10 kb clones will be chosen from this data set, and these will be sequenced to completion by a shotgun approach. The group plans to annotate the sequence extensively and make it freely available to the public on a weekly basis. The rate of sequencing will be about 25kb per week, and the direct cost rate will be less than $0.30 per finished base.
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