Chaper 1 : Sequencing Microbial Genomes

Abstract:

This chapter gives an overview of the strategies developed to sequence entire microbial genomes, and discusses the advantages and disadvantages of various approaches. For total-genome shotgun sequencing, the genomic DNA is fragmented into random pieces and subcloned directly into pUC, Ml3, or other vectors that accept insert sizes of 1 to 5 kbp. Typically, 6 to 10 genome equivalents are sequenced to cover the DNA molecule completely by using standard primers that prime at the end of the cloning vector. The primer-walking strategy has been tried primarily in the context of the yeast sequencing project. The method requires an ordered library of clones, either an overlapping set of large clones (e.g., a cosmid library) or an ordered set of discrete subclones (e.g., two 6-base cutter restriction digest libraries from a cosmid). Regardless of the sequencing strategy chosen in a particular project, there are four general phases of the sequencing process. They are primary sequencing phase, linking phase, polishing phase, and finished sequence. Only one genome project, the Escherichia coli effort at the University of Wisconsin, made substantial progress with radioactive sequencing before changing to automated-sequencing strategies. There are two different kinds of sequencing laboratories that produce genomic sequence: sequencing factories and smaller laboratories with an output of 2 to 5 Mbp of genomic sequence per year. With increasing levels of automation, the sequence production costs will be reduced, and in the future it may be possible to reach 10 cents per finished base pair.