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Generation of Sequencing Libraries for the Pneumocystis Genome Project

The Pneumocystis Genome project was initiated in April 1999 with the goal of sequencing the genome of Pneumocystis carinii [3,4]. The original strategy for sequencing was to create a physical map from an 8–103 cosmid library and to use this minimum tile as the source of DNA for shotgun libraries [1]. Shotgun libraries were to be constructed with nebulized cosmid DNA, which would then be cloned into a blunt-end vector and sequenced. Problems resulting from this approach included the sequencing of unacceptable amounts of the pWEB cosmid vector DNA; significant contamination of the sequencing libraries with bacterial host DNA; and a cloning bias of some regions of the P. carinii genome. A new strategy for sequencing the P. carinii genome utilizing a modified vector and sticky-end cloning with subsequent southern hybridization-based screening of inserts was found to provide a higher percentage of assembled sequence than the previous blunt-end strategy and is described.