Soil Microbial DNA Purification Strategies for Multiple Metagenomic Applications

Parsley, Larissa C.; Wu, Chun; Mead, David A.; Goodman, Robert M.; Liles, Mark R.

doi:10.1002/9781118010549.ch11

Cited by 1 publication

(1 citation statement)

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“…Similar to fosmid cloning, the present method facilitates potential reconstruction of entire viral “genomes" due to the deliberately-reduced sequence complexity in the sample. Fosmid cloning, however, is subject to cloning-related artifacts such as counterselection against potentially toxic genes often found in phage genomes, bias toward capture of specific-size inserts, influence of modified DNA often present in viral genomes, and the need for high concentration, molecular weight and quality of viral DNA [55] , [56] . In addition, the generation and maintenance of a fosmid library with sufficient coverage of a complex viral community would be extremely challenging.…”

Section: Discussionmentioning

confidence: 99%

Finding a Needle in the Virus Metagenome Haystack - Micro-Metagenome Analysis Captures a Snapshot of the Diversity of a Bacteriophage Armoire

et al. 2012

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Viruses are ubiquitous in the oceans and critical components of marine microbial communities, regulating nutrient transfer to higher trophic levels or to the dissolved organic pool through lysis of host cells. Hydrothermal vent systems are oases of biological activity in the deep oceans, for which knowledge of biodiversity and its impact on global ocean biogeochemical cycling is still in its infancy. In order to gain biological insight into viral communities present in hydrothermal vent systems, we developed a method based on deep-sequencing of pulsed field gel electrophoretic bands representing key viral fractions present in seawater within and surrounding a hydrothermal plume derived from Loki's Castle vent field at the Arctic Mid-Ocean Ridge. The reduction in virus community complexity afforded by this novel approach enabled the near-complete reconstruction of a lambda-like phage genome from the virus fraction of the plume. Phylogenetic examination of distinct gene regions in this lambdoid phage genome unveiled diversity at loci encoding superinfection exclusion- and integrase-like proteins. This suggests the importance of fine-tuning lyosgenic conversion as a viral survival strategy, and provides insights into the nature of host-virus and virus-virus interactions, within hydrothermal plumes. By reducing the complexity of the viral community through targeted sequencing of prominent dsDNA viral fractions, this method has selectively mimicked virus dominance approaching that hitherto achieved only through culturing, thus enabling bioinformatic analysis to locate a lambdoid viral “needle" within the greater viral community “haystack". Such targeted analyses have great potential for accelerating the extraction of biological knowledge from diverse and poorly understood environmental viral communities.

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Section: Discussionmentioning

confidence: 99%