Viromes, Not Gene Markers, for Studying Double-Stranded ... · umvirate of gene, population, and...
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Viromes, Not Gene Markers, for Studying Double-Stranded DNA Virus Communities Matthew B. Sullivan Department of Ecology and Evolutionary Biology, University of Arizona, Tucson Arizona, USA Microbes have recently been recognized as dominant forces in nature, with studies benefiting from gene markers that can be quickly, informatively, and universally surveyed. Viruses, where explored, have proven to be powerful modulators of locally and globally important microbes through mortality, horizontal gene transfer, and metabolic reprogramming. However, community- wide virus studies have been challenged by the lack of a universal marker. Here, I propose that viral metagenomics has advanced to largely take over study of double-stranded DNA viruses. T here was a time, not so long ago, when exploring diversity in wild viral communities required researchers to focus on gene markers. Because viruses do not share a single gene, such gene markers can target only specific viral groups, including the T4-like myoviruses (e.g., major capsid and portal proteins), T7-like podo- viruses (e.g., DNA polymerase), and phycodnaviridae (e.g., DNA polymerase). Gene marker studies complemented fluorescence or electron microscopy-based counts of virus-like particles to help advance the field one step closer to considering the details of natural viral community diversity and interactions. By assessing genetic variability in these target groups, clear links were made between diversity and how it changes over space and time, partic- ularly by leveraging throughput to examine high-resolution tem- poral dynamics in concert with parallel measurements for mi- crobes (see, e.g., reference 1). Here, I propose that it is time to experimentally evaluate whether amplicon-derived operational taxonomic unit abun- dances correlate with their actual abundances in nature. Why do we need to evaluate this if microbial ecologists routinely conduct similar gene marker studies? Unlike our microbial counterparts, for whom nondegenerate primers commonly exactly match com- munity sequences, viral ecologists are often forced to employ highly degenerate primer sets designed from insufficient databases and low PCR annealing temperatures (details appear in reference 2). Even with fewer concerns, microbial ecologists also remain conservative in using such data only in unweighted analyses, thereby implicitly acknowledging that absolute abundances might be significantly altered by the PCR process in ways yet undocu- mented. Such a practice should perhaps also become mainstream in viral ecology. Fortunately, the sample-to-sequence viral meta- genome (virome) pipeline, at least for the double-stranded DNA (dsDNA) viruses that pass through a 0.2-m filter, offers a means to experimentally evaluate the quantitative performance of PCR-based studies. Further, drawing upon examples from the study of oceanic viral communities, I suggest that, with the right experimental design, viromics offers advantages over the study of gene markers for infer- ring biology in complex systems and testing a priori and ad hoc hy- potheses (summarized in Fig. 1 and detailed below). Why is viromics not yet mainstream? First, sampling for envi- ronmental viruses often yields too little material for standard se- quencing libraries. However, numerous groups have now shown that sequencing libraries can be made from far less than 1 nano- gram of DNA (3–5) and that linker-amplified viromes are quan- titative (1.5-fold) from so little DNA (4). Second, unknown sequences dominate viromes, as 63 to 93% of the reads often lack functional or taxonomic annotations (reviewed in reference 6). However, new approaches now illumi- nate this “viral dark matter.” For example, coverage of viral-ge- nome sequence space is rapidly improving through genome se- quencing of isolates in culture collections that represent abundant and rare ocean viruses (see, e.g., references 7, 8, and 9), as well as through extraction of sequence data of novel viruses from single-cell genomics projects (10). Further, smaller viral genomes are being as- sembled from marine metagenomes (see, e.g., references 11 and 10), and strategies to simplify more-complex viral communities are en- abling assembly of large genomic regions of larger viruses (12). Today, deeper sequencing combined with extrapolation of “population-level” variability from wild T4-like phage genome variability (12) enables ocean researchers to identify and quantify populations in a virome even when a database representative is completely lacking. One study (10) mined 186 microbial and viral metagenomes to show that uncultivated SUP05 viruses are persis- tent and evolutionarily dynamic over 3 years but endemic to the particular study site. Further, the genomic context for these new SUP05 viruses suggested that they manipulate the central and de- fining metabolism of their SUP05 bacterial hosts through virus- encoded sulfur-cycling genes (10), a feature shared with a prior study of viruses assembled from microbiomes (11). Thus, viromes can simultaneously map the spatiotemporal variation of many target groups while also providing genome-enabled hypotheses about eco- logical drivers of any particular target group. This is, of course, limited to those populations that are abundant in the data set, but as sequenc- ing improves, many populations already qualify. Two other approaches have emerged to help viral ecologists make meaningful inferences from virome “dark matter.” Protein Accepted manuscript posted online 24 December 2014 Citation Sullivan MB. 2015. Viromes, not gene markers, for studying double- stranded DNA virus communities. J Virol 89:2459 –2461. doi:10.1128/JVI.03289-14. Editor: C. S. Sullivan Address correspondence to [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03289-14 March 2015 Volume 89 Number 5 jvi.asm.org 2459 Journal of Virology on May 19, 2015 by UNIVERSITY OF ARIZONA LIBRARY http://jvi.asm.org/ Downloaded from
Transcript of Viromes, Not Gene Markers, for Studying Double-Stranded ... · umvirate of gene, population, and...
Viromes, Not Gene Markers, for Studying Double-Stranded DNAVirus Communities
Matthew B. Sullivan
Department of Ecology and Evolutionary Biology, University of Arizona, Tucson Arizona, USA
Microbes have recently been recognized as dominant forces in nature, with studies benefiting from gene markers that can bequickly, informatively, and universally surveyed. Viruses, where explored, have proven to be powerful modulators of locally andglobally important microbes through mortality, horizontal gene transfer, and metabolic reprogramming. However, community-wide virus studies have been challenged by the lack of a universal marker. Here, I propose that viral metagenomics has advancedto largely take over study of double-stranded DNA viruses.
There was a time, not so long ago, when exploring diversity inwild viral communities required researchers to focus on gene
markers. Because viruses do not share a single gene, such genemarkers can target only specific viral groups, including the T4-likemyoviruses (e.g., major capsid and portal proteins), T7-like podo-viruses (e.g., DNA polymerase), and phycodnaviridae (e.g., DNApolymerase). Gene marker studies complemented fluorescence orelectron microscopy-based counts of virus-like particles to helpadvance the field one step closer to considering the details ofnatural viral community diversity and interactions. By assessinggenetic variability in these target groups, clear links were madebetween diversity and how it changes over space and time, partic-ularly by leveraging throughput to examine high-resolution tem-poral dynamics in concert with parallel measurements for mi-crobes (see, e.g., reference 1).
Here, I propose that it is time to experimentally evaluatewhether amplicon-derived operational taxonomic unit abun-dances correlate with their actual abundances in nature. Why dowe need to evaluate this if microbial ecologists routinely conductsimilar gene marker studies? Unlike our microbial counterparts,for whom nondegenerate primers commonly exactly match com-munity sequences, viral ecologists are often forced to employhighly degenerate primer sets designed from insufficient databasesand low PCR annealing temperatures (details appear in reference2). Even with fewer concerns, microbial ecologists also remainconservative in using such data only in unweighted analyses,thereby implicitly acknowledging that absolute abundances mightbe significantly altered by the PCR process in ways yet undocu-mented. Such a practice should perhaps also become mainstreamin viral ecology. Fortunately, the sample-to-sequence viral meta-genome (virome) pipeline, at least for the double-stranded DNA(dsDNA) viruses that pass through a 0.2-�m filter, offers a means toexperimentally evaluate the quantitative performance of PCR-basedstudies. Further, drawing upon examples from the study of oceanicviral communities, I suggest that, with the right experimental design,viromics offers advantages over the study of gene markers for infer-ring biology in complex systems and testing a priori and ad hoc hy-potheses (summarized in Fig. 1 and detailed below).
Why is viromics not yet mainstream? First, sampling for envi-ronmental viruses often yields too little material for standard se-quencing libraries. However, numerous groups have now shownthat sequencing libraries can be made from far less than 1 nano-
gram of DNA (3–5) and that linker-amplified viromes are quan-titative (�1.5-fold) from so little DNA (4).
Second, unknown sequences dominate viromes, as �63 to93% of the reads often lack functional or taxonomic annotations(reviewed in reference 6). However, new approaches now illumi-nate this “viral dark matter.” For example, coverage of viral-ge-nome sequence space is rapidly improving through genome se-quencing of isolates in culture collections that represent abundantand rare ocean viruses (see, e.g., references 7, 8, and 9), as well asthrough extraction of sequence data of novel viruses from single-cellgenomics projects (10). Further, smaller viral genomes are being as-sembled from marine metagenomes (see, e.g., references 11 and 10),and strategies to simplify more-complex viral communities are en-abling assembly of large genomic regions of larger viruses (12).
Today, deeper sequencing combined with extrapolation of“population-level” variability from wild T4-like phage genomevariability (12) enables ocean researchers to identify and quantifypopulations in a virome even when a database representative iscompletely lacking. One study (10) mined 186 microbial and viralmetagenomes to show that uncultivated SUP05 viruses are persis-tent and evolutionarily dynamic over 3 years but endemic to theparticular study site. Further, the genomic context for these newSUP05 viruses suggested that they manipulate the central and de-fining metabolism of their SUP05 bacterial hosts through virus-encoded sulfur-cycling genes (10), a feature shared with a priorstudy of viruses assembled from microbiomes (11). Thus, viromescan simultaneously map the spatiotemporal variation of many targetgroups while also providing genome-enabled hypotheses about eco-logical drivers of any particular target group. This is, of course, limitedto those populations that are abundant in the data set, but as sequenc-ing improves, many populations already qualify.
Two other approaches have emerged to help viral ecologistsmake meaningful inferences from virome “dark matter.” Protein
Accepted manuscript posted online 24 December 2014
Citation Sullivan MB. 2015. Viromes, not gene markers, for studying double-stranded DNA virus communities. J Virol 89:2459 –2461. doi:10.1128/JVI.03289-14.
Editor: C. S. Sullivan
Address correspondence to [email protected].
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.03289-14
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clustering organizes viral sequence space into arbitrarily definedbins that can be quantified. Such PC-based “gene ecology” ad-vances include establishing (i) a sampling effort and communitydiversity (6), (ii) core gene sets as windows into the functions(known and unknown) required across viral communities (13),and (iii) flexible gene sets that define niche-differentiating func-tions across viral communities (13). Additionally, new system-level phenomena can be unveiled. For example, it now appearsthat vertical vectoring of viral particles from the surface to thedepths of oceans strongly influences the genetic diversity of deep-sea viral communities (13). Yet, these powerful PC-based ap-proaches are only part of the picture, as currently they map only�70% of virome reads and are dependent upon front-end assem-bly and gene prediction. To remedy this, kmer abundances can bederived from every virome read and, when coupled to networkingalgorithms, used to develop ecological models that explore driversof viral-community niche differentiation (14). Both areas are en-abling new scientific discovery, development, and application,and when combined with population analyses, they lead to a tri-
umvirate of gene, population, and community ecology at a sys-tem-wide level (reviewed in reference 15).
Finally, experimentally linking viruses (and associated vi-romes) to host cells is now possible through viral tagging (12).This enables exploration of viral-genome sequence space in asso-ciation with particular cultivable host cells. When this process iscoupled with other recently developed virus-host linkage methods(e.g., phageFISH, microfluidic digital PCR, fosmids, and SAGs[reviewed in reference 15]), it is clear that viral ecologists have anemerging toolkit for evaluating virus-host interaction dynamics atunprecedented spatiotemporal scales and for specific virus-hostpairings—all as virome-enabled alternatives to PCR-based genemarker studies.
Placing these advances aside, the reality is that two additionalroadblocks keep viromics from widespread use. First, user-friendly tools and extensive databases for analyzing and interpret-ing viromes are underdeveloped compared to their microbialcounterparts. Fortunately, new tools are emerging; they includeMetaVir (16) and VIROME (17), which offer Web-based inter-
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Niche Differentiation 13
Identify photic and aphotic viruses
SUP05 viruses 16
Persistent, evolutionarily
dynamic viruses endemic to OMZs
ANALYTICAL ADVANCES
K-mer Network Analysis 14
Determining ecological parameters that drive community structure
ECOLOGICAL CONTEXTNEW VIRAL BIOLOGY
SAR11 91st most abundant
marine phage
100 nm
Cellulophaga phages 7
Ubiquitous, rare marine phages
SAR116 8 2nd most abundant
marine phage
Deep Water Vectoring 13
Vertical flux of viruses from photic to aphotic
zone
Global Virome Size 20
Re-estimated to a few million proteins
Metabolic Reprogramming 19
Viral gene families potential to reprogram metabolic flux through
central pathways
Protein Clusters 6Viral proteins in ecological and evolutionary context
Databases 10, 17
Web interfaces foranalyzing viromes
ENABLED TECHNIQUES
Viral Tagging 12
Links viruses to specific host cells for high-throughput
screening and sequencing
FIG 1 Viromics is now optimized for studying dsDNA viruses in nature. Enabled by an optimized sample-to-sequence pipeline (4) and analytical advances (6,13, 14), databases and tools (16, 17), and quantitative viromes and virome-enabled techniques (12) are now revealing significant new biological information fornatural viral communities (13, 19, 20) and ecological context for newly isolated and genome-sequenced (7–9) or virome-determined viruses (10). POV, PacificOcean viromes; OMZs, oxygen minimum zone.
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faces for analyzing and interpreting viromes and large and grow-ing publicly available virome databases. Complementarily, iVirus(http://ivirus.us/) is a recent effort leveraging the iPlant cyberin-frastructure to a point where a user can access cutting-edge, high-performance computing capability without computer scienceknowledge. Moreover, this platform enables users to design andmake their own tools available to the community using iPlantcomputational resources and an application programming inter-face. New viromic “apps” (software programs), curated data sets,and query-able metadata are now being made accessible in iPlant(http://www.iplantcollaborative.org/) through the iVirus/iMi-crobe project using either a graphical user interface or commandline interface, depending upon user capabilities (http://www.moore.org/grants/list/GBMF4491). These emerging resourcesmean that interpreting viromes is getting easier. The second bot-tleneck is cost. I will simply suggest that at some point, with plum-meting sequencing costs, viromes will become a more cost-effec-tive means to document viral-community structure than genemarkers, at least for abundant viral populations. Arguably, if re-searchers consider personnel and reagent costs for primer devel-opment, optimization, and analyses for PCR-based studies andweigh the relative differences in the resulting biological inferencesthat can be made from the data, then that time is now. If not now,then it will be soon, likely as major community-available infor-matics tools and databases mature.
Undoubtedly, PCR-based gene marker studies remain critical(i) where viromics remains poorly developed (e.g., with single-stranded DNA [ssDNA] and RNA viruses and with dsDNA virusesof �0.2 �m), (ii) for targeted studies for which gene markers aremined from quantitative viromes to assess population-level spa-tiotemporal variability (see, e.g., reference 18), and (iii) wherehigh-resolution data sets for a target viral population are neededto answer a particular research question (see, e.g., reference 1).However, for the smaller dsDNA viral communities, I suggest thatviromes are largely ready to quantitatively evaluate and likely re-place PCR-based gene marker surveys to address most of the fun-damental questions needing answers in viral ecology.
ACKNOWLEDGMENTS
Grants from the Gordon and Betty Moore Foundation (2631, 3790), aswell as Tucson Marine Phage Laboratory members past and present, areacknowledged for enabling method developments that led to the quanti-tative sample-to-sequence pipeline for �0.2-�m dsDNA viruses.
Bonnie Hurwitz, Simon Roux, and Eric Wommack are thanked fortheir development of viromics analytical tools, and Christine Schirmer isthanked for preparing Fig. 1.
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