GENOME SEQUENCING ON BOTH STRANDS - THE JANUS STRATEGY

The design of large scale DNA sequencing projects such as genome analy sis demands a new approach to sequencing strategy, since neither a pur ely random nor a purely directed method is satisfactory. We have devel oped a strategy that combines these two methods in a way that preserve s the advantages of both while avoiding their particular limitations. Computer simulations showed that a specific balance of random and dire cted sequencing was required for the most efficient strategy, termed t he Janus strategy, which has been used in the Escherichia coli genome sequencing project. This approach depended on obtaining sequence easil y from either strand of a cloned insert, and was facilitated by invers ion of the insert in the engineered M13 vector Janus, by site-specific recombination. The inversion was accomplished simply by growth on the appropriate host strain, when the DNA strand incorporated into the ne w single stranded phage was complementary to that in the original phag e, and was sequenced by the same simple protocol as the first strand.