FINE-MAPPING OF SHOTGUN TEMPLATE-LIBRARIES - AN EFFICIENT STRATEGY FOR THE SYSTEMATIC SEQUENCING OF GENOMIC DNA

To test the effectiveness of ordering shotgun DNA-templates prior to s equence analysis, the 450 kb left arm of yeast chromosome XII was rand omly subcloned into a phagemid vector. Clones were ordered by hybridis ation to an average map density of one new insert every 125 bp and are currently used for sequencing the chromosomal fragment. An 11.5 kb ov erlap between the template map and a DNA fragment that had been sequen ced earlier allowed an independent evaluation of the strategy's effect iveness. To this end, clones were selected from the map and tag-sequen ced from either end, thus comparing the map position with the actual l ocation within the 11.5 kb. Of 65 selected clones, taken mostly at ran dom from a total of 423, 58 mapped on average about a quarter of a clo ne length around their predicted position, with the other seven being between 0.6 and 1.5 clone length off. 75-86 sequencing reactions on cl ones selected from the map would have been sufficient for completely s equencing both strands of the 11.5 kb fragment. The results demonstrat e the efficacy of such template sorting, considerably assisting sequen cing at relatively little cost on the mapping level.