Ordered shotgun sequencing, a strategy for integrated mapping and sequencing of YAC clones.
Ordered shotgun sequencing proposes to organize the mapping and sequencing of YACs with a hierarchical strategy that incorporates a feedback loop. Building on current protocols, a YAC is subcloned into plasmids, plasmid insert ends are sequenced, and the sequences are overlapped to create a partial map. Complete sequencing then starts with plasmids whose end-sequence tracts have overlapped, but to a minimal extent. The next plasmids to be sequenced are again selected for least overlap, striking out progressively to span the YAC with minimal directed gap-filling. Simulations support its feasibility and indicate that during the generation of the complete sequence, the approach facilitates the early choice of regions for selective sequencing, for example, for coding units. The sequencing of plasmids would also require less redundancy, and discriminate repetitive sequences more easily, than random sequencing across larger clones. The overall effort scales with YAC size and can be further reduced by additional mapping information.
Chen
E Y
EY
Advanced Center for Genetic Technology, Applied Biosystems, Inc., Foster City, California 94404.
Schlessinger
D
D
Kere
J
J
D002874:Chromosome Mapping / Q000379:methods*; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D003198:Computer Simulation; D005069:Evaluation Studies as Topic; D015894:Genome, Human; D006801:Humans; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D016324:Sequence Tagged Sites