This project will research a large-scale DNA sequencing strategy based on multiplex DNA sequencing of ordered priming sites on cosmid-sized templates derived from human cosmid contigs and sequentially-derived cosmids from human yeast artificial chromosomes. The regions of interest chosen for sequencing include the NF1 locus from chromosome 17, and the familial polyposis region on chromosome 5. A team of six people will be assembled, and their objective will be to test methodologies for megabase sequencing currently under development here in Utah. Their task will be to fractionate YACs into ordered cosmids, to saturate these cosmids with transposon-based multiplex priming sites, to determine the minimal-spanning set of these priming sites, to batch process these clones through template preparation, sequencing and electrophoresis using a multiplex protocol, to recover the data by blotting and sequential probing with multiplex identifiers, to implement an automated film-reader capable of accurate base-calling, and to complete the sequence by employing merging, error-correcting and gap-filling routines. A specific advantage given to this group is its incorporation within a setting where areas including multiplex sequencing, capillary gel electrophoresis, film-reading algorithms and informatic issues of data and laboratory management are being actively researched. By the end of the third year, the production rate of completed sequence from this group is projected to be in the 1 megabase-pair (Mb)/year range. The goal of the research effort will be to provide a robust test for the developing technology and a realistic evaluation of the cost of acquiring contiguous human genomic sequence at the Mb level.
