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Resources » Paper

Harris BR et al. (1997) International C. elegans Meeting "Sequencing C. elegans YACs"

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    Status:
    Publication type:
    Meeting_abstract
    WormBase ID:
    WBPaper00022347

    Harris BR, Woessner JP, & Consortium TCeGS (1997). Sequencing C. elegans YACs presented in International C. elegans Meeting. Unpublished information; cite only with author permission.

    In order to obtain the maximum information in the shortest time, C. elegans sequencing was initiated within the gene-rich regions of the genome, which are estimated to contain over 80% of genes. Encompassing 60 MB, this region also had the advantage of being well represented in cosmid contigs. Sequencing of this 60 Mb region is now essentially complete. Of the remaining sequence approximately 20 Mb is only represented by YAC clones. Thus it was imperative that new sequencing strategies were developed so that YACs could be utilised as part of the initial sequencing substrate. During 1996, production scale methodologies were established in both labs to allow a shotgun sequencing approach to be efficiently applied to YAC clones. The major difficulties are handling the small amount of DNA that can be recovered from yeast hosts and elimination of yeast contamination. However, by double gel purification of YAC isolates, and the use of yeast 'window' strains where necessary, yeast sequence contamination can be reduced below 10%. pUC is better than M13 for providing adequate libraries from small amounts of DNA. (The Sanger Centre uses exclusively pUC, while the Genome Sequencing Center uses a mixture of pUC and M13). The pUC clones have the additional advantages of bridging inverted repeats (which M13 cannot handle) and of providing an extensive check on assembly from forward and reverse read pairs. Our strategy for sequencing the final 40 Mb will be to shotgun sequence YAC clones in conjunction with any underlying cosmid clones. This strategy is proceeding well and at the time of writing more than 40 YAC clones are being sequenced. We estimate that approximately 150 YACs will need to be shotgunned to complete the genomic sequence.

    Affiliation:
    - The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, UK CB101RQ Washington University School of Medicine, Genome Sequencing Center, St. Louis, MO 63108


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