Abstract
Metagenomic analyses of marine viruses generate an overview of viral genes present in a sample, but the percentage of the resulting sequence fragments that can be reassembled is low and the phenotype of the virus from which a given sequence derives is usually unknown. In this study, we employed physical fractionation to characterize the morphological and genomic traits of a subset of uncultivated viruses from a natural marine assemblage. Viruses from Kāne‘ohe Bay, Hawai‘i were fractionated by equilibrium buoyant density centrifugation in a cesium chloride (CsCl) gradient, and one fraction from the CsCl gradient was then further fractionated by strong anion-exchange chromatography. One of the fractions resulting from this two-dimensional separation appeared to be dominated by only a few virus types based on genome sizes and morphology. Sequences generated from a shotgun clone library of the viruses in this fraction were assembled into significantly more numerous contigs than have been generated with previous metagenomic investigations of whole DNA viral assemblages with comparable sequencing effort. Analysis of the longer contigs (up to 6.5 kb) assembled from our metagenome allowed us to assess gene arrangement in this subset of marine viruses. Our results demonstrate the potential for physical fractionation to facilitate sequence assembly from viral metagenomes and permit linking of morphological and genomic data for uncultivated viruses.
Highlights
Viruses are the most abundant biological entities in aquatic environments and have significant roles that include causing mortality, mediating genetic exchange, and altering the genetic potential of their hosts [1]
Investigations of the morphology and genome size distributions [3] of aquatic viruses have shown that they are a diverse component of aquatic ecosystems
Viral Fractionation In the initial continuous cesium chloride (CsCl) gradient of the viral concentrate, a large portion of the viruses banded with little resolution over a broad range and at high densities (1.47–1.56 g ml21; data not shown), an atypical result for this method [23]
Summary
Viruses are the most abundant biological entities in aquatic environments and have significant roles that include causing mortality, mediating genetic exchange, and altering the genetic potential of their hosts [1]. Investigations of the morphology (reviewed by [2]) and genome size distributions [3] of aquatic viruses have shown that they are a diverse component of aquatic ecosystems. Investigating the genomic content of this diverse array of viruses has proven to be challenging. Isolation of viruses from cultivated hosts allows for the sequencing of complete viral genomes which can be used to connect genomic with phenotypic information (e.g., [4,5]) and to determine the gene organization and genetic capabilities of a given virus (e.g., [4,6]). It has been estimated that .99% of environmental microorganisms are uncultivated [7] and that the groups of microorganisms that are in culture may not be representative of the environments from which they originate [8]
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