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Monterey Bay Coastal
Ocean Microbial Observatory |
| Random Shotgun Sequencing and Closure |
Image ©MBARI |
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Introduction
Large-insert closure sequencing of environmentally derived BACs is valuable tool for characterizing the biological properties of uncultivated microorganisms. Individual BAC clones are handles in the same manner are whole genomes in term of sequencing and assembly. In fact they are typically much easier than a whole microbial genome because of the smaller size.
Single celled marine picoplankton are the most abundant, biogeochemically important organisms in the oceans. Beyond bulk abundance and estimates, the qualitative attributes and activities of individual components of these microbial communities are crucial to ecosystem function. It is well established that many central biogeochemical processes including nitrogen fixation, anaerobic respiration, chemolithoautotrophy, and nitrification, are mediated solely by prokaryotic species. The cycling of the elements in the sea is critically dependent on such microbial activities, yet in the marine environment the specific microbes mediating these processes, and their genetic and biochemical properties, are poorly characterized or unknown.
Molecular ecological surveys of PCR amplified rRNA genes have revealed unsuspected phylogenetic diversity contained within natural microbial populations. It is apparent that some newly discovered, uncultivated bacteria and archaea represent major components of natural marine microbial populations. Despite the impact of these rRNA-based gene surveys, phylogenetic identification based solely on rRNA sequence does not allow inference of physiology, biochemistry, and ecological significance. Therefore, the specific biological properties of these abundant uncultivated microorganisms therefore remain almost entirely unknown.
Methods
BAC closure sequencing. One small insert (2-3 kb) plasmid library was generated by mechanical shearing of genomic DNA. Briefly, purified BAC DNA is nebulized for 1 min at 20 psi. The sheared DNA is size fractionated on an agarose gel, blunt-ended, ligated into pUC derived vectors, and transformed into Escherichia coli by electorporation. Plasmids are purified from transformed colonies, and the plasmid inserts sequenced using ABIPrisim 3700 capillary sequencers. Approximately 8-fold coverage is attained for each BAC, and the sequences are assembled using the TIGR Assembler. Primer walking on plasmid clones closes sequence gaps. Physical gaps were closed by multiplexed and combinatorial PCR followed by sequencing of the PCR products obtained.
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