Sequence Assembly and Finishing

Sequence Assembly: The pUC18 sequences will be assembled at each site to evaluate the quality of sequencing. All participating centers will receive copies of sequence assembly tools and quality control assessment software, PHRED/PHRAP and Consed to perform initial assembly and to add their raw sequence reads to the central sequencing data repository at the Fungal Genome Database. Initially assembled data will also be transferred to the Assembly Center at the University of Kentucky, Dr. Charles Staben, Director, for final editing and to generate the strategy for closure. Within 24 hours, all assembled contigs greater than 2 kbp would be submitted to Genbank as Level 1 data. Gaps and closures will be filled in by investigators at the University of Cincinnati using 4 strategies: 1) Ends of contigs will be hybridized to the clone collection to close gaps. With this strategy, slow but steady progress can be made to eliminate contig and cell boundaries (i.e. collections of neighboring clones linked together by shared hybridizations to common clones). 2) Southern hybridization of probes at the ends of cells (and contigs) to genomic DNA digested with restrictions enzymes, e.g. SmaI, SstII to link cells will be performed. 3) The ends of identified contigs will be linked by NotI digestions of whole chromosomes. Probes will be prepared from the ends of contigs. These probes will be hybridized to NotI fragments of the whole chromosomes. Contigs linked to common NotI fragments fall on the same NotI fragment. This approach is less labor intensive than producing a NotI restriction map, which is one of the common closure methods of connecting contigs . 4) Outward facing primers from the sequenced ends of probes at the ends of contigs) that bracket a gap will be used to create PCR products spanning small and medium sized gaps. A final strategy would be to take a probe at the end of a cell (or contig) and prepare successively smaller disjoint pools of primers from the ends of other cells. Then the neighboring end, which provides the other primer for the PCR product, can be found by a "divide and conquer" strategy.