Since its inception in 1990, our collaborative effort to sequence the C. elegans genome has continually increased throughput. Our basic strategy uses shotgun sequencing of cosmids followed by directed reads for gaps and seqeunce ambiguities. Greater efficiency has been achieved through the use of modified protocols, new sequencing chemistries, mechanization and automation of specific steps and importantly, new software tools. Currently, we have finished 38 Mb of sequence and have more than 20 Mb of incomplete, but assembled sequence, covering almost all of the gene-rich portions of the autosomes and all of the X chromosome. About 20% of the genome, located primarily on the chromosomal arms, is not represented in cosmids and is present only in YACs. In addition, there are small regions in the gene-rich portions where there are no mapped cosmids or the representative full-length cosmid cannot be recovered. We are developing two strategies for obtaining this remaining sequence: 1) screening of a C. elegans genomic library made in the fosmid vector pFOS1, and 2) subcloning YAC DNA for shotgun sequencing. The fosmid vector is single copy and the C. elegans fosmids tend to be less prone to deletion than cosmids. The fosmid library has been gridded onto high density filter arrays (18,000 clones) and is being hybridized with YAC DNA and PCR-derived probes adjacent to gaps. Fosmids yielding a positive signal are subjected to fingerprinting analysis to determine the best representatives for subsequent sequencing. The C. elegans fosmids, like the cosmids, do not appear to cover all of the chromosome arms and thus the YACs containing these regions remain the only available cloned source. To refine the map positions of the YACs and to confirm their integrity, we are testing the feasibility of performing restriction digests. Based on this information, YACs will be selected, purified by pulse field gel electrophoresis and subcloned into
m13 vectors for our standard shotgun sequencing. Analysis and assembly of these large YAC sequencing projects will be facilitated by having the sequence of the underlying cosmids/fosmids in hand.