Optimized multiplex PCR: Efficiently closing a whole-genome shotgun sequencing project

A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory proce dures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR p rimers are used in a single sequencing reaction, is used to interpret the m ultiplex PCR results. Two protocols are presented, one that minimizes pipet ting and another that minimizes the number of reactions, The pipette optimi zed multiplex PCR method has been employed in the final phases of closing t he Streptococcus pneumoniae genome sequence, with excellent results. (C) 19 99 Academic Press.