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The Sanger sequencing method produces relatively long DNA sequences of unmatched quality and has been considered for long time as the gold standard for sequencing DNA. Many improvements of the Sanger method that culminated with fluorescent dyes coupled with automated capillary electrophoresis enabled the sequencing of the first genomes. Nevertheless, using this technology to sequence whole genomes was costly, laborious and time consuming even for genomes that are relatively small in size. A major technological advance was the introduction of next-generation sequencing (NGS) pioneered by 454 Life Sciences in the early part of the 21th century. NGS allowed scientists to sequence thousands to millions of DNA molecules in a single machine run. Since then, new NGS technologies have emerged and existing NGS platforms have been improved, enabling the production of genome sequences at an unprecedented rate as well as broadening the spectrum of NGS applications. The current affordability of generating genomic information, especially with microbial samples, has resulted in a false sense of simplicity that belies the fact that many researchers still consider these technologies a black box. In this review, our objective is to identify and discuss four steps that we consider crucial to the success of any NGS-related project. These steps are: (1) the definition of the research objectives beyond sequencing and appropriate experimental planning, (2) library preparation, (3) sequencing and (4) data analysis. The goal of this review is to give an overview of the process, from sample to analysis, and discuss how to optimize your resources to achieve the most from your NGS-based research. Regardless of the evolution and improvement of the sequencing technologies, these four steps will remain relevant.

The following protocol is used to construct shotgun libraries from RNA virus assemblages. This method is designed to detect all RNA viruses, regardless of their genome orientation, and therefore provides a broader assessment of RNA virus diversity compared to the single-gene–based method described in "A degenerate primer reverse transcriptionpolymerase chain reaction–based protocol to determine the diversity of picorna-like viruses."

This protocol is a minor modification of that reported by Culley and Steward (2007). As the starting point for this protocol, we assume that viruses have been collected on an aluminumoxide 0.02-µm syringe-tip filter (Anotop, Whatman), but other filters capable of capturing viruses may be substituted. This is a protocol from: Steward, G. F. and A. I. Culley. 2010. Chapter 16: Extraction and purification of nucleic acids from viruses.Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7 Please see the published manuscript for additional information.

The traditional phenol extraction procedure, based on early work by Kirby (1957) and elaborated in most modern protocol compendia, yields very clean nucleic acids suitable for a variety of downstream applications. This extraction method is coupled with a routine alcohol precipitation step to allow buffer exchange, removal of trace amounts of chloroform, and concentration of nucleic acids (Sambrook and Russell 2001). This is a protocol from: Steward, G. F. and A. I. Culley. 2010. Chapter 16: Extraction and purification of nucleic acids from viruses.Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7 Please see the published manuscript for additional information.

The diversity of ribonucleic acid (RNA) viruses in the ocean and the ongoing isolation and characterization of RNA viruses that infect important primary producers suggests that RNA viruses are active members of the marine microbial assemblage. At this point, little is known about the composition, dynamics, and ecology of the RNA virioplankton. In this protocol, we describe a method to assess RNA virus diversity from seawater.

If one wishes to have a stock of high molecular weight viral DNA that can be stored for long periods of time with minimal shearing or degradation, the viruses can be embedded in agarose before extraction. Extraction of embedded cells is the standard procedure for sizing the genomes of bacteria andyeast by PFGE (Sambrook and Russell 2001), and a similar protocol can be used for embedded viruses (Rohozinski et al. 1989; Lanka et al. 1993; Wommack et al. 1999; Sandaa et al. 2010, this volume). This is a protocol from: Steward, G. F. and A. I. Culley. 2010. Chapter 16: Extraction and purification of nucleic acids from viruses.Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7 Please see the published manuscript for additional information.

Early work on marine algal viruses focused exclusively on those having DNA genomes, but recent studies suggest that RNA viruses, especially those with positive-sense, single-stranded RNA (+ssRNA) genomes, are abundant in tropical and temperate coastal seawater. To test whether this was also true of polar waters, we estimated the relative abundances of RNA and DNA viruses using a mass ratio approach and conducted shotgun metagenomics on purified viral samples collected from a coastal site near Palmer Station, Antarctica on six occasions throughout a summer phytoplankton bloom (November-March). Our data suggest that RNA viruses contributed up to 65% of the total virioplankton (8-65%), and that, as observed previously in warmer waters, the majority of RNA viruses in these Antarctic RNA virus metagenomes had +ssRNA genomes most closely related to viruses in the order Picornavirales. Assembly of the metagenomic reads resulted in five novel, nearly complete genomes, three of which had features similar to diatom-infecting viruses. Our data are consistent with the hypothesis that RNA viruses influence diatom bloom dynamics in Antarctic waters.

Heterosigma akashiwo (Rhaphidophyceae) is a unicellular, flagellated, bloom-forming, toxic alga of ecological and economic importance. Here, we report the results of sequencing and analyzing the genome of an 8.6-kb single-stranded RNA virus (HaRNAV-SOG263) that infects H. akashiwo. Our results show that HaRNAV is related to picorna-like viruses, but does not belong within any currently defined virus family. This is based on the genome organization and sequence comparisons of putative RNA-dependent RNA polymerase (RdRp), helicase, and capsid protein sequences. The genome sequence predicts a single open reading frame (orf) encoding a polyprotein that contains conserved picorna-like protein domains, with putative nonstructural protein domains present in the N-terminus and the structural proteins in the C-terminus of the polyprotein. We have analyzed and compared the virus structural proteins from infectious and noninfectious particles. In this way, we identified structural protein cleavage sites as well as protein processing events that are presumably important for maturation of virus particles. The combination of genome structure and sequence relationships to other viruses suggests that HaRNAV is the first member of a proposed new virus family (Marnaviridae), related to picorna-like viruses.

Picorna-like viruses are a loosely defined group of positive-sense single-stranded RNA viruses that are major pathogens of animals, plants and insects. They include viruses that are of enormous economic and public-health concern and are responsible for animal diseases (such as poliomyelitis), plant diseases (such as sharka) and insect diseases (such as sacbrood). Viruses from the six divergent families (the Picornaviridae, Caliciviridae, Comoviridae, Sequiviridae, Dicistroviridae and Potyviridae) that comprise the picorna-like virus superfamily have the following features in common: a genome with a protein attached to the 5' end and no overlapping open reading frames, all the RNAs are translated into a polyprotein before processing, and a conserved RNA-dependent RNA polymerase (RdRp) protein. Analyses of RdRp sequences from these viruses produce phylogenies that are congruent with established picorna-like virus family assignments; hence, this gene is an excellent molecular marker for examining the diversity of picorna-like viruses in nature. Here we report, on the basis of analysis of RdRp sequences amplified from marine virus communities, that a diverse array of picorna-like viruses exists in the ocean. All of the sequences amplified were divergent from known picorna-like viruses, and fell within four monophyletic groups that probably belong to at least two new families. Moreover, we show that an isolate belonging to one of these groups is a lytic pathogen of Heterosigma akashiwo, a toxic-bloom-forming alga responsible for severe economic losses to the finfish aquaculture industry, suggesting that picorna-like viruses are important pathogens of marine phytoplankton.

HaRNAV, a novel virus that infects the toxic bloomforming alga Heterosigma akashiwo (Hada) Hada ex Hada et Chihara, was characterized based on morphology, pathology, nucleic acid type, structural proteins, and the range of host strains that it infects. HaRNAV is a 25-nm single-stranded RNA (ssRNA) virus with a genome size of approximately 9100 nucleotides. This is the first report of an ssRNA virus that causes lysis of a phytoplankton species. The virus particle is sensitive to chloroform and contains at least five structural proteins ranging in apparent size from 24 to 34 kDa. HaRNAV infection causes swelling of the endoplasmic reticulum and progeny virus particles assemble in the cytoplasm of the host, frequently in crystalline arrays. The infectivity of HaRNAV was tested against 15 strains of H. akashiwo isolated from Japanese waters, the Northeast Pacific, and the Northwest Atlantic. HaRNAV caused lysis of three strains from the Northeast Pacific and two strains from Japan but none from the Northwest Atlantic. The characterization of HaRNAV demonstrates that HaRNAV is a novel type of phytoplankton virus but has some similarities with plant viruses belonging to the Sequiviridae and to other known ssRNA viruses. Further genomic analysis, however, is necessary to determine any phylogenetic relationships. The discovery of HaRNAV emphasizes the diversity of H. akashiwo viral pathogens and, more importantly, algal‐virus pathogens and the complexity of virus‐host interactions in the environment.

Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses.

Viruses have a profound influence on the ecology and evolution of plankton, but our understanding of the composition of the aquatic viral communities is still rudimentary. This is especially true of those viruses having RNA genomes. The limited data that have been published suggest that the RNA virioplankton is dominated by viruses with positive-sense, single-stranded (+ss) genomes that have features in common with those of eukaryote-infecting viruses in the order Picornavirales (picornavirads). In this study, we investigated the diversity of the RNA virus assemblages in tropical coastal seawater samples using targeted PCR and metagenomics. Amplification of RNA-dependent RNA polymerase (RdRp) genes from fractions of a buoyant density gradient suggested that the distribution of two major subclades of the marine picornavirads was largely congruent with the distribution of total virus-like RNA, a finding consistent with their proposed dominance. Analyses of the RdRp sequences in the library revealed the presence of many diverse phylotypes, most of which were related only distantly to those of cultivated viruses. Phylogenetic analysis suggests that there were hundreds of unique picornavirad-like phylotypes in one 35-liter sample that differed from one another by at least as much as the differences among currently recognized species. Assembly of the sequences in the metagenome resulted in the reconstruction of six essentially complete viral genomes that had features similar to viruses in the families Bacillarna-, Dicistro-, and Marnaviridae. Comparison of the tropical seawater metagenomes with those from other habitats suggests that +ssRNA viruses are generally the most common types of RNA viruses in aquatic environments, but biases in library preparation remain a possible explanation for this observation., IMPORTANCE Marine plankton account for much of the photosynthesis and respiration on our planet, and they influence the cycling of carbon and the distribution of nutrients on a global scale. Despite the fundamental importance of viruses to plankton ecology and evolution, most of the viruses in the sea, and the identities of their hosts, are unknown. This report is one of very few that delves into the genetic diversity within RNA-containing viruses in the ocean. The data expand the known range of viral diversity and shed new light on the physical properties and genetic composition of RNA viruses in the ocean.

Viruses are an integral component of the marine food web, contributing to the disease and mortality of essentially every type of marine life, yet the diversity of viruses in the sea, especially those with RNA genomes, remains very poorly characterized. Isolates of RNA-containing viruses that infect marine plankton are still rare, and the only cultivation-independent surveys of RNA viral diversity reported so far were conducted for temperate coastal waters of British Columbia. Here, we report on our improvements to a previously used protocol to investigate the diversity of marine picorna-like viruses and our results from applying this protocol in subtropical waters. The original protocol was simplified by using direct filtration, rather than tangential flow filtration, to harvest viruses from seawater, and new degenerate primers were designed to amplify a fragment of the RNA-dependent RNA polymerase gene by reverse transcription-PCR from RNA extracted from the filters. Whereas the original protocol was unsuccessful in a preliminary test, the new protocol resulted in amplification of picorna-like virus sequences in every sample of subtropical and temperate coastal seawater assayed. These polymerase sequences formed a diverse, but monophyletic cluster along with other sequences amplified previously from seawater and sequences from isolates infecting marine protists. Phylogenetic analysis suggested that our sequences represent at least five new genera and 24 new species of RNA viruses. These results contribute to our understanding of RNA virus diversity and suggest that picorna-like viruses are a source of mortality for a wide variety of marine protists.

Background RNA viruses have been isolated that infect marine organisms ranging from bacteria to whales, but little is known about the composition and population structure of the in situ marine RNA virus community. In a recent study, the majority of three genomes of previously unknown positive-sense single-stranded (ss) RNA viruses were assembled from reverse-transcribed whole-genome shotgun libraries. The present contribution comparatively analyzes these genomes with respect to representative viruses from established viral taxa. Results Two of the genomes (JP-A and JP-B), appear to be polycistronic viruses in the proposed order Picornavirales that fall into a well-supported clade of marine picorna-like viruses, the characterized members of which all infect marine protists. A temporal and geographic survey indicates that the JP genomes are persistent and widespread in British Columbia waters. The third genome, SOG, encodes a putative RNA-dependent RNA polymerase (RdRp) that is related to the RdRp of viruses in the family Tombusviridae, but the remaining SOG sequence has no significant similarity to any sequences in the NCBI database. Conclusion The complete genomes of these viruses permitted analyses that resulted in a more comprehensive comparison of these pathogens with established taxa. For example, in concordance with phylogenies based on the RdRp, our results support a close homology between JP-A and JP-B and RsRNAV. In contrast, although classification of the SOG genome based on the RdRp places SOG within the Tombusviridae, SOG lacks a capsid and movement protein conserved within this family and SOG is thus likely more distantly related to the Tombusivridae than the RdRp phylogeney indicates.

Viruses of marine bacteria (bacteriophage) have been characterized since the mid-20th century (1). However, it took several additional decades before electron micrographs of seawater revealed that particles of virus-like morphology could exceed concentrations of 1 million per milliliter of seawater (2, 3), and that phage infection could cause substantial mortality in marine bacterial communities (4). Subsequent research in the field of marine viral ecology has focused on the characterization of the diversity of the virioplankton (community of extracellular viruses), elucidating how viruses influence the community dynamics and evolution of their hosts and ultimately the role viruses play in biogeochemical cycling. Critical to the understanding of viral ecology is the identification of the constituents of the viral community and whom they infect. This is a formidable challenge because there are thousands of different types of viruses in every liter of seawater (5) that are mostly distantly related to the catalog of known viruses, and whose morphology and genotype do not identify the host that they infect. A clear, albeit challenging, path forward to gaining a greater understanding of viral ecology is the isolation and characterization of viruses that infect hosts who play important roles in the environment. In PNAS, Kang et al. (6) make a significant contribution to the field with a report describing the isolation and characterization of a bacteriophage, HMO-2011, which infects a member of the SAR116 clade, a group of marine bacteria that are abundant and … [↵][1]2E-mail: aculley{at}hawaii.edu. [1]: #xref-corresp-1-1

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.

Investigating the bacteriophages of vibrios has led to significant insights into the evolution and pathogenicity of their host strains. This report presents the first complete genome sequence of a bacteriophage that infects the deadly human pathogen Vibrio vulnificus. The phage was isolated from the surface waters of the Ala Wai Canal, which is part of an urban watershed in eastern Honolulu, Hawaii, USA. The phage particle is icosahedral, with a diameter of 35-40 nm, and a small non-contractile tail. The genome was sequenced in its entirety, rendering a 38 kb sequence located on a single, linear, circularly permuted chromosome. Here, we present the annotation and genomic features of the bacteriophage, VvAW1.