Phage Metagenomics

Abstract

The vast majority of novel DNA sequences deposited in the databases now comes from environmental phage DNA sequences. Methods are presented for the cloning and sequencing of phage DNA that might otherwise be lethal to bacterial host vectors or contain modified DNA bases that prevent standard cloning of such sequences. In addition, methods are presented for the isolation of viral particles directly from soil and sediment environmental samples or from large volumes of environmental water samples. The viral particles are then purified by cesium-chloride density centrifugation followed by DNA extraction. This purified viral metagenomic DNA is then used for cloning and sequencing.

Direct Assessment of Viral Diversity in Soils by Random PCR Amplification of Polymorphic DNA

“…Prior to analysis by RAPD-PCR, viral concentrates were treated with a high concentration of DNase I to remove free, extraviral DNA (39). Five units of DNase I (RQ1 RNase-free DNase; Fisher Scientific, Pittsburg, PA), 2 l of 10ϫ DNase buffer, and 20 l of viral concentrate or viral extract were mixed, and the mixture was incubated at 37°C for 30 min.…”

Genomic Characterization of Mycobacteriophage Giles: Evidence for Phage Acquisition of Host DNA by Illegitimate Recombination

“…The complete genome sequences of approximately 400 double-stranded DNA tailed phages have been determined, which show, together with viral metagenomic studies (1,5,8), that the phage population has very high genetic diversity and is replete with genetic information that either is very distant from other known sequences or has a distinct evolutionary origin. Furthermore, the completely sequenced phage genomes have characteristic mosaic architectures, so that modules, composed of either single genes or several genes, are shared between individual genomes (6,15,18,28); each phage genome can thus be considered a unique assemblage of individual modules.…”

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis

“…DNA was extracted from eight different samples taken over a 2 year period (Table 1) Viral DNA was extracted from seawater and sponge phage concentrates via the procedure described by Casas & Rohwer (2007). DNA concentration was measured using a PerkinElmer Victor 2 Fluorometer and analysed by using PerkinElmer WorkOut 2.5 Data analysis software.…”