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FEMS Microbiology Letters, 363, 2016, fnw027 doi: 10.1093/femsle/fnw027 Advance Access Publication Date: 5 February 2016 Minireview M I N I R E V I E W – Virology Single-stranded DNA phages: from early molecular biology tools to recent revolutions in environmental microbiology Anna J. Székely1,2,∗,† and Mya Breitbart2 1 Department of Ecology and Genetics/Limnology, Uppsala University, Uppsala 752 36, Sweden and 2 College of Marine Science, University of South Florida, St Petersburg 33701, FL, USA ∗ Corresponding author: Norbyvägen 18 D, Uppsala 752 36, Sweden. Tel: +46 18 4712796; E-mail: [email protected] One sentence summary: In light of the recent recognition of the high diversity and cosmopolitan distribution of single-stranded DNA phages, this manuscript presents an overview of the major differences between single-stranded and double-stranded DNA phages that may influence their effects on bacterial communities and make their simultaneous study in environmental samples challenging. Editor: Andrew Millard † Anna J. Székely, http://orcid.org/0000-0001-5764-2141 ABSTRACT Single-stranded DNA (ssDNA) phages are profoundly different from tailed phages in many aspects including the nature and size of their genome, virion size and morphology, mutation rate, involvement in horizontal gene transfer, infection dynamics and cell lysis mechanisms. Despite the importance of ssDNA phages as molecular biology tools and model systems, the environmental distribution and ecological roles of these phages have been largely unexplored. Viral metagenomics and other culture-independent viral diversity studies have recently challenged the perspective of tailed, double-stranded DNA (dsDNA) phages, dominance by demonstrating the prevalence of ssDNA phages in diverse habitats. However, the differences between ssDNA and dsDNA phages also substantially limit the efficacy of simultaneously assessing the abundance and diversity of these two phage groups. Here we provide an overview of the major differences between ssDNA and tailed dsDNA phages that may influence their effects on bacterial communities. Furthermore, through the analysis of 181 published metaviromes we demonstrate the environmental distribution of ssDNA phages and present an analysis of the methodological biases that distort their study through metagenomics. Keywords: ssDNA phages; Microviridae; Inoviridae; viral metagenomics; methodological biases INTRODUCTION Early on in the study of viruses capable of killing bacteria (i.e. bacteriophages or phages), the first single-stranded DNA (ssDNA) phage φX174 was isolated from the sewers of Paris (Sertic and Bulgakov 1935). Though the nature of its genome was not understood until decades later (Sinsheimer 1959), the fact that this virus maintained infectivity even after passing through the smallest pore-size filters available at the time indicated the extremely small size of this phage compared to other isolates. In the early 1960s, the Ff phages, a distinct group of filamentous ssDNA phages infecting conjugative Escherichia coli strains (i.e. Fpilus positive strains), were also isolated from sewage systems in Europe and the USA (Loeb 1960; Hofschneider 1963; Marvin and Hoffmann-Berling 1963). The small, circular ssDNA genomes of φX174 and the Ff phages led to their prominent roles as model systems and tools in the rise and golden era of molecular biology. Among many other milestones, φX174 was the first DNA genome to be sequenced (Sanger, Nicklen and Coulson 1977) and the Received: 19 November 2015; Accepted: 2 February 2016 C FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected] 1 2 FEMS Microbiology Letters, 2016, Vol. 363, No. 6 Figure 1. Hierarchical taxonomic composition of (a) phage species recognized by the ICTV (b) and sequence reads identified as phages in metaviromes deposited in Metavir (Roux et al. 2014b). For the generation of (b), only previously published metaviromes with unassembled reads available were included in order to be able to verify the methods of the library generation (total = 181; Table S1, Supporting Information). Taxonomic composition of the reads was computed by Metavir by performing BLASTx comparison with the NCBI Refseq complete viral genomes protein sequence database (release of 2015-01–05). The reads were genome length normalized using GAAS (Angly et al. 2009) and only reads that presented a significant hit (threshold of 50 on the BLAST bitscore) were included. first genome to be constructed in vitro from synthesized oligonucleotides (Smith et al. 2003), while the Ff phages, especially M13, have been used for cloning (Messing et al. 1977), phage display (Smith 1985) and nanotechnology (Finnigan et al. 2004). For most of the past century, the discovery of novel phages remained dependent on classical plaque assay techniques using cultured bacterial hosts. As a result, the majority of phages in the latest taxonomy release from the International Committee on Taxonomy of Viruses (ICTV) (Adams et al. 2015), were isolated on bacterial hosts belonging to relatively easily culturable groups. In addition, most of the ICTV-recognized phages are tailed, double-stranded DNA (dsDNA) phages belonging to the order Caudovirales (Fig. 1a). Despite the importance of ssDNA phages in the development of molecular biology and their heavy exploitation for biotechnological purposes, ssDNA phages only represent 11% of the phages in the ICTV database. The majority of these are filamentous phages belonging to the family Inoviridae, including the Ff phages described above, while the family Microviridae, which includes φX174, only represents 2% of the database. Based on the ICTV database, the electron microscopic analysis of cultured phages (Ackermann 2007) and genome size analysis of environmental viral genomes (Wommack et al. 1999), the general paradigm for many years has been that the vast majority of the phages are tailed dsDNA phages. Advances in molecular biology revealed the vast diversity of environmental bacteria that could not be cultured using standard laboratory methods (Pace et al. 1986) foreshadowing the severe underestimation of phage diversity represented by cultured isolates (Breitbart, Miyake and Rohwer 2004). Ironically, although molecular biology techniques developed through the use of phages, the lack of universal phage genes such as the riboso- mal RNA genes found in cellular organisms substantially limits the application of molecular techniques for studying phage diversity. Numerous studies have utilized amplification of ‘signature genes’, which are conserved among limited members of specific phage groups to study the diversity of uncultured phages (Adriaenssens and Cowan 2014); however, it was the introduction of viral metagenomics in 2002 that first shed light on the vast diversity of viruses in the environment (Breitbart et al. 2002). Subsequent advances in the field of viral metagenomics enabled the recovery of ssDNA viruses (Angly et al. 2006) and RNA viruses (Culley, Lang and Suttle 2006) as well. Viral metagenomic studies have provided significant insight regarding the diversity, ecology, genetic content, depth distribution and biogeography of dsDNA phages (Rosario and Breitbart 2011). Less research has addressed the prevalence and ecological roles of ssDNA phages or compared the distributions of these two DNA phage types. This knowledge gap is significant since the ratio of ssDNA phages to dsDNA phages among the published metavirome libraries deposited in Metavir, a web server designed to annotate viral metagenomic datasets (Roux et al. 2014b), is more than double of the ratio in the ICTV database (Fig. 1b). Furthermore, most of the ssDNA phages in the metaviromes are related to members of the family Microviridae, which is typically regarded as a group of specialized phages that only infect a narrow range of hosts (Enterobacteria and intracellular parasites such as Chlamydia) (Cherwa and Fane 2011). However, the generation of viral metagenomic datasets has many known (Kim and Bae 2011) and potential biases, which leads to uncertainties regarding the true abundance and importance of ssDNA phages. Therefore, the scope of this review is to present the following issues in regards to their relevance for the study of ssDNA Székely and Breitbart 3 phages in environmental samples: (i) the main aspects in which ssDNA phages differ from tailed, dsDNA phages (Caudovirales) and (ii) the prevalence of ssDNA phages in prior environmental studies and current limitations to their study. HOW DIFFERENT ARE ssDNA PHAGES FROM TAILED dsDNA PHAGES? Phages with both DNA and RNA genomes have been described; however, only a few RNA phage isolates exist and RNA phages appear to be very rare even in metagenomic surveys specifically targeting viral RNA (Djikeng et al. 2009; Rosario et al. 2009). Similarly, though several non-tailed dsDNA phage families are recognized by ICTV (Adams et al. 2015), the number of isolates and the abundance of these phages in metaviromes (Fig. 1b) are significantly less than that of the tailed dsDNA phages. The low recovery of RNA and non-tailed dsDNA phages from metaviromes may result from the inability of most computational tools to recognize similar phages due to the low number of reference sequences. Due to the limited amount of available data, this study focuses on comparing ssDNA phages to tailed dsDNA phages. Furthermore, as detailed reviews about the morphology, genome structure and life cycle of both tailed phages (Ackermann 2007) and ssDNA phages (Cherwa and Fane 2011; Rakonjac 2012; MaiProchnow et al. 2015) already exist, here we focus on the differences that may affect the ecology of these groups and the efficacy of their study using metagenomic methods. Phylogeny and taxonomy Based on their ability to infect both bacteria and archaea and similarities in the three-dimensional structure of their capsid, all tailed phages are classified into a single monophyletic order (Caudovirales) within dsDNA viruses (Ackermann 2007). In contrast, ssDNA viruses are currently classified into eight families without order-level affiliation. Two of the eight ssDNA virus families infect bacteria: the Microviridae family, which is comprised of ssDNA phages with small icosahedral capsids (including φX174) and the Inoviridae family, which contains the filamentous ssDNA phages (including the Ff phages) (Fig. 2). Although these two ssDNA phage families differ significantly in their morphology, they share a key genomic feature since members of both families contain small circular genomes that are replicated through rolling-circle replication (RCR) (Krupovic 2013). The RCR initiation proteins of ssDNA viruses cluster with plasmid-encoded RCR genes, which gave rise to the hypothesis that although ssDNA viruses are polyphyletic, they likely emerged through a similar mechanism when plasmids using RCR acquired virion structural modules from distinct sources to yield ssDNA viruses (Krupovic 2013). Although ssDNA phages are likely polyphyletic, each of the two families is unquestionably monophyletic. Members of the Microviridae family all have small (∼25 nm) icosahedral capsids with a T = 1 triangulation number (Table 1). The family can be divided into two phylogenetic lineages: the genus Microvirus and the subfamily Gokushovirinae (Fig. 2) (Brentlinger et al. 2002; Roux et al. 2012). A distinguishing characteristic between the two lineages is the 12 spikes on the 5-fold vertices of the Microvirus icosahedral capsid. These spikes are built by the major spike protein (G protein in φX174), which is separate from the major capsid protein (MCP; F protein in φX174) that forms the main subunits of the icosahedron (Bennett, McKenna and AgbandjeMcKenna 2008). In contrast, the Gokushovirinae lack spikes at the Figure 2. Classification, phylogeny and host range of the (a) Microviridae and (b) Inoviridae families. Taxonomic and phylogenetic group names are bold and host names are indicated with bullet points. Taxonomic groups that have not yet been proposed to or confirmed by ICTV are in apostrophes. Proposed hosts not verified by cultivation are in parentheses. The clades of the genus Microvirus are named after representative phages. The genus Inovirus is divided into classes based on X-ray fiber diffraction patterns. ∗ phylogenetic lineage, not ICTV approved taxonomic classification. † hosts suggested based on integrated prophage genomes. ‡ hosts suggested based on co-occurrence. ∗ ∗ host suggested based on single-cell genomics association. § hosts of not yet ICTV approved inoviruses. 5-fold vertices, but instead have ‘mushroom-like’ protrusions at the 3-fold vertices of the icosahedrons (Cherwa and Fane 2011), which are formed by an insertion loop encoded within a hypervariable region of the MCP (Chipman et al. 1998; Roux et al. 2012; Hopkins et al. 2014). In the current ICTV taxonomy release (Adams et al. 2015), the family Microviridae contains the Microvirus genus (which is not in a subfamily) and the subfamily Gokushovirinae, which contains three genera (Bdellomicrovirus, Chlamydiamicrovirus, Spiromicrovirus; Fig. 2a). Additional viral genomes reconstructed from metagenomic libraries have led to the recognition of further phylogenetic clusters within the Microviridae family; however, these clusters have not been formally proposed or accepted by the ICTV to date. For the Microvirus lineage a new clade (candidate genus Pequeñovirus) has been suggested based on genomes discovered in methane seep sediments (Bryson et al. 2015). Similarly, two more subfamilies have been suggested for the Gokushovirinae lineage: Alpavirinae (Krupovic and Forterre 2011), which seem to be mainly associated with human microbiota and Pichovirinae, which have a broader environmental range (Roux et al. 2012). Both of these newly proposed subfamilies have the mushroom-like protrusion encoded within their MCP gene similar to Gokushovirinae. Members of the Inoviridae family have long (700–2000 nm), thin, filamentous capsids composed of thousands of major 4 FEMS Microbiology Letters, 2016, Vol. 363, No. 6 Table 1. Summary of the main differences between tailed dsDNA phages and the two ssDNA phage families. Genome type Genome size (kb) Mutation rate (substitution/nucleotide/infection) Virion morphology Triangulation number of icosahedron Diameter (nm) Length (nm) Buoyant density (g cm−3 ) Caudovirales Inoviridae Microviridae References Linear dsDNA 16–500 9.8 × 10−8 (T2) Circular ssDNA 4.5–12.4 7.9 × 10−7 (M13) Circular ssDNA 4.4–6.1 1.1 × 10−6 (φX174) King et al. (2012) King et al. (2012) Sanjuán et al. (2010) Head–tail structure 3–52 (head) Filamentous – Icosahedral 1 King et al. (2012) King et al. (2012) 45–170 (head) up to 230 (elongated head) 3–825 (tail) 6 (Inovirus) 10–15 (Plectrovirus) 700–3700 (Inovirus) 230–280 (Plectrovirus) 1.28 (Inovirus) ∼25 – King et al. (2012) Cherwa and Fane (2011) King et al. (2012) 1.30–1.31 (Gokushovirinae) King et al. (2012) 1.39 (Plectrovirus) 1.4 (Spiromicrovirus) 1.38–1.41 (Microvirus) Lytic (host-genome integration detected, lysogeny mechanism unknown) 1.46–1.54 (1.37 only φKZ) Infection mode Lytic or lysogenic Chronic infection Cell lysis Holins and peptidoglycan hydrolases – capsid protein subunits (Marvin, Symmons and Straus 2014). The ICTV classifies members of this family into two genera: the thinner filament-like Inovirus and the thicker rod-shaped Plectrovirus (Table 1, Fig. 2b). Virion length is correlated to genome size for both genera; however, plectoviruses are several-fold shorter relative to their genome length than inoviruses. Both the length and diameter differences between these two genera are related to differences in ssDNA packing and structure (Mai-Prochnow et al. 2015). Inoviruses can be further classified based on their X-ray fiber diffraction pattern into two classes: Class I, which contains the Ff phages and other inoviruses that infect E. coli, and Class II, which contains the inoviruses that infect other Gram-negative bacteria. However, many inovirus isolates still remain uncharacterized by this method (Fig. 2) (Marvin, Symmons and Straus 2014). Host range Lack of knowledge regarding the bacterial targets of these ssDNA phages presents the ultimate conundrum in deciphering their ecological effects. While definitive linkage between phages and their hosts is still mostly dependent on culturing techniques, the emergence of new techniques such as identification of prophages within bacterial genomes (Krupovic and Forterre 2011) and metagenomes (Waller et al. 2014), the analysis of CRISPR spacers within potential hosts (Andersson and Banfield 2008), co-occurrence patterns based on relative abundances in environmental samples (Bryson et al. 2015), and single-cell level tools (Dang and Sullivan 2014) and analyses (Roux et al. 2014a) are continuously expanding our understanding of phage host range. While the Caudovirales contains both bacteriophages and archaeal viruses (Prangishvili, Forterre and Garrett 2006); members of the Microviridae and Inoviridae families are only known to infect bacteria and some of the ssDNA phage lineages are be- Peptidoglycan synthesis inhibitors (φX174) Ackermann (2007) Mai-Prochnow et al. (2015) Krupovic and Forterre (2015) Ackermann (2007) Bernhardt et al. (2002) lieved to have quite narrow host ranges (Fig. 2). For example, all cultured phages belonging to the Microvirus genus were isolated on members of the Enterobacteriaceae family. However, the related pequeñoviruses were recently identified in methane seeps where Enterobacteriaceae are absent (Bryson et al. 2015). Additionally, these phages display differences in the genes encoding the capsid spike-forming protein believed to be responsible for host adherence, suggesting that this Microvirus lineage infects different hosts. All cultured representatives of the Gokushovirinae lineage were isolated from intracellular parasites (Fig. 2a), so for many years it was hypothesized that gokushoviruses are specialized to the niche of intracellular hosts, where competition with other phages is limited (Cherwa and Fane 2011). However, the recent identification of gokushoviruses associated with single-cell genomes of Gammaproteobacteria (Roux et al. 2014a) and the discovery of gokushoviruses integrated into Bacteroidetes genomes (candidate subfamily Alpavirinae) (Krupovic and Forterre 2011; Roux et al. 2012) demonstrates that the host range of these phages is not limited to intracellular parasites. The first members of the Inoviridae (Ff phages) were also isolated from Enterobacteria and all of the ICTV approved species of the genus Inovirus infect Gammaproteobacteria (Fig. 2) (Day 2012). However, additional phages related to inoviruses (not yet ICTV-approved) have been discovered infecting Betaproteobacteria, Thermus and Gram-positive Firmicutes (Pederson et al. 2001; Chopin et al. 2002; Bille et al. 2005; Yamada et al. 2007). Similar to the gokushoviruses, members of the Plectrovirus genus have only been isolated from intracellular parasites that lack cell walls (Gourlay 1970). Life cycle, infection mode and effects on host Though the basic steps of the lytic infection cycle are the same for all phages, the studied members of the two families of Székely and Breitbart ssDNA phages differ in the initial steps of infection (i.e., particle attachment, adsorption and DNA entry) (Ackermann 2007; Cherwa and Fane 2011; Rakonjac 2012). However, once inside the bacterial cell, if genome integration does not occur, the studied members of the Inoviridae and Microviridae follow similar strategies for replication and transcription. The main difference in these processes compared to tailed dsDNA phages results from their distinct genomic material and the size of their genomes; the smaller genome size of ssDNA phages forces them to rely more heavily upon the molecular machinery of their host to produce new phages. From an ecological point of view, it is also important to note that the two ssDNA phage families differ greatly from each other and from the dsDNA tailed phages in regard to viral particle release. The dsDNA tailed phages encode complex cell lysis machineries including proteins that induce membrane lesions (holins) and peptidoglycan hydrolases, which allow them to precisely time virion release in order to maximize fitness (Wang 2005). In contrast, members of the Microviridae, which have a much more limited coding capacity, only encode a single protein (protein E in φX174, product of OrfN in MH2K) (Cherwa and Fane 2012) that—according to studies of φX174 cell lysis mechanism— inhibits cell wall synthesis and induces lysis in a manner similar to a penicillin peptidoglycan synthesis inhibitor (Bernhardt et al. 2002). As a result, φX174 also lyses its hosts, though the timing of lysis seems to depend solely on the growth rate of the host, while fitness adjustment happens through the modification of infection rates (Roychoudhury et al. 2013). In contrast, filamentous phages do not lyse their hosts during virion release, but instead continuously shed viral particles causing ‘stable’ or ‘chronic’ infections (Mai-Prochnow et al. 2015). This process, except for some ‘superinfective’ variants, does not kill the host, though the production of viral particles does induce bacterial stress-response processes (Rakonjac 2012). Another difference between the infection modes of these phage groups is the frequency of lysogeny. Following DNA entry, many tailed phages are maintained in a vegetative multiplication state (i.e. lysogeny) by integrating into the host genome or persisting as plasmids within the bacterial cytoplasm (Ackermann 2007; Payet and Suttle 2013). Inoviridae regularly integrate into their hosts’ genomes using phage-encoded integrases, host recombinases or in the case of some plectroviruses, phage-encoded transposases (Krupovic and Forterre 2015). However, in contrast to the tailed phages, the integrated filamentous prophages continue to produce and shed viral particles through the chronic infection process (Mai-Prochnow et al. 2015). Prior to the recent discovery of prophage sequences related to members of the Microviridae within bacterial genomes, this phage group was not thought to undergo lysogeny. However, recent results suggest the existence of such a process (Krupovic and Forterre 2011), though the lack of integrases or transposases in the genomes of members of the Microviridae family suggests utilization of a host recombinase-based mechanism similar to that of some inoviruses (Krupovic and Forterre 2015). Finally, although both ssDNA and dsDNA tailed phages continuously undergo changes through mutations and horizontal gene transfer (HGT), some marked differences in these processes exist between the phage types. Both ssDNA and dsDNA phages follow Drake’s rule, which assumes that DNA-based entities have equal mutation rates per replication per genome (Drake 1991; Cuevas, Duffy and Sanjuán 2009). However, due to the small genome size of ssDNA phages, Drake’s rule leads to higher mutation rates per base per infection than for dsDNA phages (Sanjuán et al. 2010). Consequently, ssDNA viruses 5 have mutation rates that are intermediate between the rapidly evolving RNA and the more slowly evolving dsDNA viruses (Duffy, Shackelton and Holmes 2008). On the other hand, HGT is much more common among dsDNA phages because their larger genomes and capsids allow more flexibility for exchange of genetic material. This genetic flexibility gives dsDNA phages the potential to augment their hosts’ physiology with properties such as virulence (Wagner and Waldor 2002) or certain metabolic capacities (Anantharaman et al. 2014). Compared to the highly mosaic dsDNA tailed phages, HGT does not appear to be as extensive among small viruses (genome <15 kb) such as the ssDNA phages (Krupovic et al. 2011). Within the Inoviridae family, the prevalence of host integration does facilitate HGT to certain extent leading to signs of previous HGT processes such as the involvement of filamentous phages in the pathogenicity of Vibrio cholerae (Faruque and Mekalanos 2003) or the substitution of the ancestral RCR genes of plectroviruses for transposase-derived versions (Krupovic 2013). Similarly, host integration has been detected in members of the Microviridae family (Krupovic and Forterre 2011) and signs of previous HGT processes have been identified for the Microvirus genus (Rokyta et al. 2006). However, the lower known prevalence of integration, combined with recent experimental evidence demonstrating fitness deficiencies of recombinant microviruses due to epistasis (Doore and Fane 2015; Sackman, Reed and Rokyta 2015), suggest that HGT is a less important process in the evolution and diversity of these ssDNA phages. ssDNA PHAGES IN ENVIRONMENTAL SAMPLES AND THE CHALLENGES TO THEIR STUDY To examine the distribution of gokushoviruses, group-specific PCR assays targeting the MCP gene have been designed based on the genomes of cultured phages and sequences recovered from metagenomic libraries. Studies conducted with these assays demonstrated the ubiquity of gokushoviruses across habitats (marine, freshwater, wastewater, sediment) and global regions (Antarctic to subtropical) (Labonté and Suttle 2013a; Hopkins et al. 2014), indicating a cosmopolitan distribution of ssDNA phages. However, the geographic distribution of individual sequences appears to be much narrower than that of dsDNA phage sequences (Desnues et al. 2008; Tucker et al. 2011; Labonté, Hallam and Suttle 2015). Similarly, analysis of metaviromes available in Metavir shows that ssDNA phage reads; especially those related to Microviridae, have been recovered from almost every habitat analyzed (Fig. 3a and b). Some habitats contained more ssDNA phages than others, but there was generally a large amount of variance in the proportion of ssDNA phages among metaviromes within a habitat. There were far fewer Inoviridae reads in every habitat, although some metaviromes from animal-associated habitats had high Inoviridae ratios (Fig. 3c). However, caution should be used in interpreting these results since we also observed differences in the recovery of ssDNA phages depending on the methods applied in generating the metaviromes. Although sequencing technologies have changed significantly over the past decade, all metaviromic studies involve the basic steps of viral concentration/purification, nucleic acid extraction and library preparation (Thurber et al. 2009). A myriad of strategies exist for generating metaviromes and although many of the basic steps are similar between studies, the order of these steps is not consistent and various studies 6 FEMS Microbiology Letters, 2016, Vol. 363, No. 6 Figure 3. Proportion of phage reads in Metavir related to (a) ssDNA phages, (b) Microviridae and (c) Inoviridae across different habitats. Each dot represents a metavirome, filled dots are metavirome generated by MDA, empty dots are metaviromes generated without MDA and the lines show the median value of the given habitat. exclude or repeat particular steps (Fig. 4). In addition, there are several alternative methods available for many of the steps (e.g. virus concentration or DNA amplification). Due to the fundamental differences between tailed dsDNA and ssDNA phages, each alternative imparts different biases on the final outcome. For example, during sample filtration and viral particle purification, the filter pore sizes may select against the largest or smallest phage groups (Table 1), the efficiency of certain methods (e.g., iron chloride precipitation) for concentrating ssDNA phages has not been tested, and chemical treatments (e.g., chloroform treatment) may differentially affect the stability of various phage groups (Thurber et al. 2009). However, the most prominent biases inducing differences between the recovery and abundance of dsDNA and ssDNA phages observed among the datasets in Metavir are the different buoyant density of the phage groups (Table 1) and whether multiple displacement amplification (MDA) was used in metavirome preparation. The two most frequently utilized methods for nonspecific amplification of viral DNA are MDA, also known as rolling circle amplification (RCA) (Angly et al. 2006) and linker amplification (LA) (Breitbart et al. 2002; Duhaime et al. 2012). Each of these methods imparts serious distortions in the ratio of dsDNA and ssDNA phages recovered. The LA method is based on the ligation of dsDNA adapters to sheared DNA (Breitbart et al. 2002; Duhaime et al. 2012). Since the ligation occurs between dsDNA fragments, ssDNA phages are not efficiently recovered by this method. On the other hand, MDA is biased towards the preferential amplification of circular ssDNA (Kim and Bae 2011). Not surprisingly the relative abundance of ssDNA phages is much higher in the metaviromes generated using MDA (Fig. 3). The preferential amplification of ssDNA viruses by MDA has even been used to specifically target ssDNA viruses in diverse metaviromic studies (Kim et al. 2008; Labonté and Suttle 2013b; Rosario et al. 2015). Fortunately, next-generation sequencing (NGS) technologies are continuously improving, requiring lower concentrations of input DNA for the preparation of metaviromic libraries and making it possible to skip the DNA amplification step (Adriaenssens et al. 2015). However, the most commonly used library preparation kits for indexing samples prior to NGS (e.g. Nextera, Illumina or ThruPLEX, Rubicon Genomics) also tag dsDNA, further hindering the possibility of gaining unbiased measurements of true phage diversity (Solonenko et al. 2013). Another property of ssDNA phages that distorts the results of metavirome analyses is their lower buoyant density compared to tailed dsDNA phages (Table 1) (Thurber et al. 2009). Cesium chloride (CsCl) gradient centrifugation is regularly used for the purification of viral particles from free nucleic acids and cellular material. Most studies describe recovering density phases above 1.35 g/ml, yet the actual density of the recovered phase is almost never reported. The presence of virus-like particles (VLP) in the recovered phase is often confirmed by nucleic acid staining and epifluorescence microscopy; however, this method is not suitable for the quantification of ssDNA phages (Holmfeldt et al. 2012). Not surprisingly, the ratio of ssDNA phages is higher in metaviromes that are generated by recovering the lower density phases after CsCl gradient centrifugation, while those generated from the highest densities (1.5 g/ml) had significantly lower ssDNA ratios despite the use of MDA (Fig. 5). Finally, it has to be noted that the vast majority of the metavirome reads from environmental samples lacks significant homology to existing databases, suggesting a great deal of viral genetic diversity has yet to be identified. For example, in the case of the metaviromes analyzed in this review, an average of only 13% of the sequences from each sample had significant similarities to reference sequences (Table S1, Supporting Information). The future exploration of this tremendous amount of genetic ‘dark matter’ has the potential to uncover additional dimensions of viral ecology that will likely include the elucidation of novel ssDNA phage diversity as well. CONCLUSIONS AND OUTLOOK The major differences between tailed phages and the two groups of ssDNA phages described here suggest that they may have Székely and Breitbart 7 Figure 4. Basic steps of viral metagenomic library generation and the strategies employed in five selected studies. a more diverse perspective including diverse ssDNA, non-tailed dsDNA and RNA phage groups. SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. FUNDING Figure 5. Proportion of phage reads related to ssDNA phages in metaviromes from different habitats in Metavir showing the strong effect of the density recovered from CsCl gradient centrifugation on the recovery of reads similar to ssDNA phages. Only metaviromes generated by MDA are presented. Each dot represents a metavirome and the lines show the median value of the given method. different ecological effects on bacterial communities. The cosmopolitan and widespread distribution of ssDNA phages, especially those related to the Gokushovirinae lineage, is evident; however, the inconsistency of protocols across studies and the methodological biases associated with the steps used to generate metaviromes limits our understanding of the true abundance and importance of ssDNA phages. The continuous improvement of sequencing techniques including the development of library preparation kits that use ssDNA as starting material (Swift Biosciences) promise to overcome the community distortion resulting from multiple displacement amplification. However, to gain a representative view of viral communities, other steps (e.g., filtration, density-dependent centrifugation) should follow consistent protocols to account for known biases. Finally, the isolation of ssDNA phages not affiliated with the two known ssDNA families (Holmfeldt et al. 2012, 2013), the dominance of non-tailed viruses in morphological surveys (Brum, Schenck and Sullivan 2013), the recognition of novel ssDNA virus clusters (Labonté and Suttle 2013b; Hopkins et al. 2014) and the expansion of single-cell sequencing for virus discovery (Yoon et al. 2011) promises to radically change our understanding of phage diversity from a view dominated by tailed phages towards This work was supported by funding received from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 330692 to AJS and grant DEB-1239976 from the U.S. National Science Foundation’s Assembling the Tree of Life Program to MB. Conflict of interest. None declared. REFERENCES Ackermann H-W. Bacteriophages: Tailed. Encycl Life Sci 2007;1–7. Adams MJ, Lefkowitz EJ, King AMQ et al. 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