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        The Genome Sequence of Haemophilus influenzae

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        Publication of the complete 1.83-Mb genome sequence of the bacterium Haemophilus influenzae strain Rd in 1995 (1 ) heralded a revolution in the study of microorganisms. The availability of a complete microbial genome sequence enormously facilitates computer-based and experimental investigations of the respective organism by providing complete lists of genes, their genetic contexts, and their predicted functions. The significance of the H. influenzae sequence was not just that it presented for the first time a complete genome from a free-living organism, but that it proved that complete genomes could be sequenced rapidly and effectively at a low cost. This was achieved through the novel approach taken by Venter and his colleagues at The Institute for Genomic Research (TIGR) to sequence the H. influenzae genome. Randomly generated small chromosomal fragments, 1–2 kb in length, were cloned into a high-copy-number plasmid vector and then both ends of the clone were sequenced using plasmid specific primers. The vast amount of DNA sequence obtained from the shotgun clones was assembled into large contiguous sequences by computers using specifically developed software. The equivalent to sequencing the entire genome 6 times was required for sufficient accuracy and coverage of the majority of the chromosome. To obtain a final contiguous sequence, gaps between contigs (tracts of contiguous sequence) were closed using homology comparisons between end sequences, the polymerase chain reaction, and sequencing of larger DNA inserts from lambda libraries. The shotgun method was rapid, as it circumvented the need for construction of a physical map of the genome, a prerequisite of the ordered approach taken to other bacterial sequencing projects.
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