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1. Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

   https://doi.org/10.7717/peerj.2777  

   Roux, Simon ; Solonenko, Natalie E. ; Dang, Vinh T. ; Poulos, Bonnie T. ; Schwenck, Sarah M. ; Goldsmith, Dawn B. ; Coleman, Maureen L. ; Breitbart, Mya ; Sullivan, Matthew B. ( December 2016 , PeerJ)

   Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).

   Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.

   Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from theMicroviridaefamily, can be among the most abundant viral genomes in a sample.

   Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

    

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2. virMine: automated detection of viral sequences from complex metagenomic samples

   https://doi.org/10.7717/peerj.6695  

   Garretto, Andrea ; Hatzopoulos, Thomas ; Putonti, Catherine ( January 2019 , PeerJ)

   Metagenomics has enabled sequencing of viral communities from a myriad of different environments. Viral metagenomic studies routinely uncover sequences with no recognizable homology to known coding regions or genomes. Nevertheless, complete viral genomes have been constructed directly from complex community metagenomes, often through tedious manual curation. To address this, we developed the software tool virMine to identify viral genomes from raw reads representative of viral or mixed (viral and bacterial) communities. virMine automates sequence read quality control, assembly, and annotation. Researchers can easily refine their search for a specific study system and/or feature(s) of interest. In contrast to other viral genome detection tools that often rely on the recognition of viral signature sequences, virMine is not restricted by the insufficient representation of viral diversity in public data repositories. Rather, viral genomes are identified through an iterative approach, first omitting non-viral sequences. Thus, both relatives of previously characterized viruses and novel species can be detected, including both eukaryotic viruses and bacteriophages. Here we present virMine and its analysis of synthetic communities as well as metagenomic data sets from three distinctly different environments: the gut microbiota, the urinary microbiota, and freshwater viromes. Several new viral genomes were identified and annotated, thus contributing to our understanding of viral genetic diversity in these three environments. 

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3. Chloroviruses

   https://doi.org/10.3390/v12010020  

   Van Etten, James L. ; Agarkova, Irina V. ; Dunigan, David D. ( January 2020 , Viruses)

   Chloroviruses are large dsDNA, plaque-forming viruses that infect certain chlorella-like green algae; the algae are normally mutualistic endosymbionts of protists and metazoans and are often referred to as zoochlorellae. The viruses are ubiquitous in inland aqueous environments throughout the world and occasionally single types reach titers of thousands of plaque-forming units per ml of native water. The viruses are icosahedral in shape with a spike structure located at one of the vertices. They contain an internal membrane that is required for infectivity. The viral genomes are 290 to 370 kb in size, which encode up to 16 tRNAs and 330 to ~415 proteins, including many not previously seen in viruses. Examples include genes encoding DNA restriction and modification enzymes, hyaluronan and chitin biosynthetic enzymes, polyamine biosynthetic enzymes, ion channel and transport proteins, and enzymes involved in the glycan synthesis of the virus major capsid glycoproteins. The proteins encoded by many of these viruses are often the smallest or among the smallest proteins of their class. Consequently, some of the viral proteins are the subject of intensive biochemical and structural investigation. 

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4. Targeted viromes and total metagenomes capture distinct components of bee gut phage communities

   https://doi.org/10.1186/s40168-024-01875-0  

   Sbardellati, Dino_Lorenzo ; Vannette, Rachel_Lee ( August 2024 , Microbiome)

   Despite being among the most abundant biological entities on earth, bacteriophage (phage) remain an understudied component of host-associated systems. One limitation to studying host-associated phage is the lack of consensus on methods for sampling phage communities. Here, we compare paired total metagenomes and viral size fraction metagenomes (viromes) as methods for investigating the dsDNA viral communities associated with the GI tract of two bee species: the European honey beeApis melliferaand the eastern bumble beeBombus impatiens.

   We find that viromes successfully enriched for phage, thereby increasing phage recovery, but only in honey bees. In contrast, for bumble bees, total metagenomes recovered greater phage diversity. Across both bee species, viromes better sampled low occupancy phage, while total metagenomes were biased towards sampling temperate phage. Additionally, many of the phage captured by total metagenomes were absent altogether from viromes. Comparing between bees, we show that phage communities in commercially reared bumble bees are significantly reduced in diversity compared to honey bees, likely reflecting differences in bacterial titer and diversity. In a broader context, these results highlight the complementary nature of total metagenomes and targeted viromes, especially when applied to host-associated environments.

   Overall, we suggest that studies interested in assessing total communities of host-associated phage should consider using both approaches. However, given the constraints of virome sampling, total metagenomes may serve to sample phage communities with the understanding that they will preferentially sample dominant and temperate phage.

    

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5. MetaPop: a pipeline for macro- and microdiversity analyses and visualization of microbial and viral metagenome-derived populations

   https://doi.org/10.1186/s40168-022-01231-0  

   Gregory, Ann C. ; Gerhardt, Kenji ; Zhong, Zhi-Ping ; Bolduc, Benjamin ; Temperton, Ben ; Konstantinidis, Konstantinos T. ; Sullivan, Matthew B. ( December 2022 , Microbiome)

   Abstract Background Microbes and their viruses are hidden engines driving Earth’s ecosystems from the oceans and soils to humans and bioreactors. Though gene marker approaches can now be complemented by genome-resolved studies of inter-(macrodiversity) and intra-(microdiversity) population variation, analytical tools to do so remain scattered or under-developed. Results Here, we introduce MetaPop, an open-source bioinformatic pipeline that provides a single interface to analyze and visualize microbial and viral community metagenomes at both the macro - and microdiversity levels. Macrodiversity estimates include population abundances and α- and β-diversity. Microdiversity calculations include identification of single nucleotide polymorphisms, novel codon-constrained linkage of SNPs, nucleotide diversity ( π and θ ), and selective pressures (pN/pS and Tajima’s D ) within and fixation indices ( F ST ) between populations. MetaPop will also identify genes with distinct codon usage. Following rigorous validation, we applied MetaPop to the gut viromes of autistic children that underwent fecal microbiota transfers and their neurotypical peers. The macrodiversity results confirmed our prior findings for viral populations (microbial shotgun metagenomes were not available) that diversity did not significantly differ between autistic and neurotypical children. However, by also quantifying microdiversity, MetaPop revealed lower average viral nucleotide diversity ( π ) in autistic children. Analysis of the percentage of genomes detected under positive selection was also lower among autistic children, suggesting that higher viral π in neurotypical children may be beneficial because it allows populations to better “bet hedge” in changing environments. Further, comparisons of microdiversity pre- and post-FMT in autistic children revealed that the delivery FMT method (oral versus rectal) may influence viral activity and engraftment of microdiverse viral populations, with children who received their FMT rectally having higher microdiversity post-FMT. Overall, these results show that analyses at the macro level alone can miss important biological differences. Conclusions These findings suggest that standardized population and genetic variation analyses will be invaluable for maximizing biological inference, and MetaPop provides a convenient tool package to explore the dual impact of macro - and microdiversity across microbial communities. 

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