Cosmid vector design for large scale sequencing: the Pyrococcus furiosus genome

Genomic sequencing can be thought of as a process of disassembling a genome into smaller pieces and subsequent reassembly back into the completed genome. This process involves the representation of the genome in libraries of DNA pieces consisting of fractions of the genome ranging in size from 100s of kilobases to hundreds of bases. In order to facilitate the process of library construction and fractionation of genomes a new cosmid vector was designed. The new cosmid vector features an inducible copy number control. Additional features that facilitate production sequencing were also included. The new vector was used in construction of a genomic library of the archea Pyrococcus furious. This library has an average insert size of 33 kb and has a genomic equivalent of 7X. This library, along with a plasmid library, was used to sequence the entire 1,908,253 bases of the P. furiosus genome. During the sequencing it was observed that stability of the cosmids and subsequent recovery of DNA were inversely proportional to the size of the insert size. Smaller inserts were more stable and had higher recovery yields than larger inserts. Further study determined that large inserts decreased the copy number of the cosmid and also inhibited the ability to induce to higher copy number. Various potential explanations were investigated.