A BAC library and paired-PCR approach to mapping and completing the genomesequence of Sulfolobus solfataricus P2

The original strategy used in the Sulfolobus solfataricus genome project wa s to sequence non overlapping, or minimally overlapping, cosmid or lambda i nserts without constructing a physical map. However, after only about two t hirds of the genome sequence was completed, this approach became counter-pr oductive because there was a high sequence bias in the cosmid and lambda li braries. Therefore, a new approach was devised for linking the sequenced re gions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end map ping and PCR screening. The PCR approaches included a novel chromosome walk ing method termed "paired-PCR". 21 gaps were filled by BAC end sequence ana lyses and 6 gaps were filled by PCR including three large ones by paired-PC R. The complete map revealed that 0.9 Mb remained to be sequenced and 34 BA C clones were selected for walking over small gaps and preparing template l ibraries for larger ones. It is concluded that an optimal strategy for sequ encing microorganism genomes involves construction of a high-resolution phy sical map by BAC end analyses, PCR screening and paired-PCR chromosome walk ing after about half the genome sequence has been accumulated.