Amplification of Uncultured Single-Stranded DNA Viruses from Rice Paddy Soil

ABSTRACT Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with φ29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.

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

PeerJ ◽

10.7717/peerj.2777 ◽

2016 ◽

Vol 4 ◽

pp. e2777 ◽

Cited By ~ 82

Author(s):

Simon Roux ◽

Natalie E. Solonenko ◽

Vinh T. Dang ◽

Bonnie T. Poulos ◽

Sarah M. Schwenck ◽

...

Keyword(s):

Multiple Displacement Amplification ◽

Ecosystem Functions ◽

Library Preparation ◽

Single Stranded Dna ◽

Preparation Methods ◽

Dna Viruses ◽

Viral Genomes ◽

Sequencing Library ◽

Dsdna Viruses ◽

Ssdna Viruses

BackgroundViruses 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).MethodsHere 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.ResultsMock 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.DiscussionTogether 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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Amplification Methods Bias Metagenomic Libraries of Uncultured Single-Stranded and Double-Stranded DNA Viruses

Applied and Environmental Microbiology ◽

10.1128/aem.00289-11 ◽

2011 ◽

Vol 77 (21) ◽

pp. 7663-7668 ◽

Cited By ~ 168

Author(s):

Kyoung-Ho Kim ◽

Jin-Woo Bae

Keyword(s):

454 Pyrosequencing ◽

Multiple Displacement Amplification ◽

Viral Diversity ◽

Viral Dna ◽

Dna Viruses ◽

Shotgun Library ◽

Amplification Method ◽

Double Stranded Dna ◽

Flow Filtration ◽

Viral Sequences

ABSTRACTInvestigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes. In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral diversity.

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DNA-spiking in viral metagenome sequencing: A new method with low bias

10.7287/peerj.preprints.2912 ◽

2017 ◽

Author(s):

Geert Cremers ◽

Lavinia Gambelli ◽

Theo van Alen ◽

Laura van Niftrik ◽

Huub Op den Camp

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Multiple Displacement Amplification ◽

Gc Content ◽

New Method ◽

Rrna Gene ◽

Natural Environments ◽

Viral Genomes ◽

Amplification Method ◽

Metagenome Sequencing

With the emergence of Next Generation Sequencing, major advances were made with regard to identifying viruses in natural environments. However, bioinformatical research on viruses is still limited because of the low amounts of viral DNA that can be obtained for analysis. To overcome this limitation, DNA is often amplified with multiple displacement amplification (MDA), which causes an unavoidable bias. Here, we describe a DNA-spiking method to avoid the bias that is created when using amplification of DNA before metagenome sequencing. To obtain sufficient DNA for sequencing, a bacterial 16S rRNA gene was amplified and the obtained DNA was spiked to a DNA sample containing DNA from a bacteriophage population before sequencing using Ion Torrent technology. After sequencing, the 16S rRNA gene reads DNA was removed by mapping to the Silva database. The new DNA-spiking method was compared with the MDA technique. When MDA was applied, the overall GC content of the reads showed a bias towards lower GC%, indicating a change in composition of the DNA sample. Assemblies using all available reads from both MDA and the DNA-spiked samples resulted in six viral genomes. All six genomes could be almost completely retrieved (97.9%-100%) when mapping the reads from the DNA-spiked sample to those six genomes . In contrast, 6.3%-77.7% of three viral genomes were covered by reads obtained using the MDA amplification method and only three were nearly fully covered (97.4%-100%). The new method provides a simple and inexpensive protocol with very low bias in sequencing of metagenomes for which low amounts of DNA are available.

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Diversity and Abundance of Single-Stranded DNA Viruses in Human Feces

Applied and Environmental Microbiology ◽

10.1128/aem.06331-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 8062-8070 ◽

Cited By ~ 136

Author(s):

Min-Soo Kim ◽

Eun-Jin Park ◽

Seong Woon Roh ◽

Jin-Woo Bae

Keyword(s):

Capsid Protein ◽

Protein Sequences ◽

Single Stranded Dna ◽

Dna Viruses ◽

Content Type ◽

Double Stranded Dna ◽

Abundance And Diversity ◽

Ssdna Viruses ◽

Virus Abundance ◽

Large Contigs

ABSTRACTIn this study, we investigated the abundance and diversity of single-stranded DNA (ssDNA) viruses in fecal samples from five healthy individuals through a combination of serial filtration and CsCl gradient ultracentrifugation. Virus abundance ranged from 108to 109per gram of feces, and virus-to-bacterium ratios were much lower (less than 0.1) than those observed in aquatic environments (5 to 10). Viral DNA was extracted and randomly amplified using phi29 polymerase and analyzed through high-throughput 454 pyrosequencing. Among 400,133 sequences, an average of 86.2% viromes were previously uncharacterized in public databases. Among previously known viruses, double-stranded DNA podophages (52 to 74%), siphophages (11 to 30%), myophages (1 to 4%), and ssDNA microphages (3 to 9%) were major constituents of human fecal viromes. A phylogenetic analysis of 24 large contigs of microphages based on conserved capsid protein sequences revealed five distinct newly discovered evolutionary microphage groups that were distantly related to previously known microphages. Moreover, putative capsid protein sequences of five contigs were closely related to prophage-like sequences in the genomes of threeBacteroidesand threePrevotellastrains, suggesting thatBacteroidesandPrevotellaare the sources of infecting microphages in their hosts.

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Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

Applied and Environmental Microbiology ◽

10.1128/aem.03878-14 ◽

2015 ◽

Vol 81 (12) ◽

pp. 3934-3945 ◽

Cited By ~ 34

Author(s):

Brian Reavy ◽

Maud M. Swanson ◽

Peter J. A. Cock ◽

Lorna Dawson ◽

Thomas E. Freitag ◽

...

Keyword(s):

Soil Types ◽

Metagenomic Sequencing ◽

Single Stranded Dna ◽

Dna Viruses ◽

Unique Type ◽

Content Type ◽

Virus Particles ◽

Intracellular Parasites ◽

Ssdna Viruses ◽

Virus Genomes

ABSTRACTThe potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the familyMicroviridae(icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis ofMicroviridaemajor coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamilyGokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar familyCircoviridaein BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in theCircoviridaeshowed that they are a novel clade ofCircoviridae-related CRESS-DNA viruses distinct from knownCircoviridaegenera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

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Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant

PeerJ ◽

10.7717/peerj.2585 ◽

2016 ◽

Vol 4 ◽

pp. e2585 ◽

Cited By ~ 11

Author(s):

Victoria M. Pearson ◽

S. Brian Caudle ◽

Darin R. Rokyta

Keyword(s):

Wastewater Treatment ◽

Microbial Communities ◽

Rolling Circle Amplification ◽

Treatment Plant ◽

Microbial Pathogens ◽

Model Organisms ◽

Single Stranded Dna ◽

Dna Viruses ◽

Depth Analysis ◽

Ssdna Viruses

Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (CircoviridaeandGeminiviridae), and model organisms for genetics and evolution studies (Microviridae). Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such asCircoviridae,Geminiviridae, andMicroviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group.

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DNA-spiking in viral metagenome sequencing: A new method with low bias

10.7287/peerj.preprints.2912v1 ◽

2017 ◽

Author(s):

Geert Cremers ◽

Lavinia Gambelli ◽

Theo van Alen ◽

Laura van Niftrik ◽

Huub Op den Camp

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Multiple Displacement Amplification ◽

Gc Content ◽

New Method ◽

Rrna Gene ◽

Natural Environments ◽

Viral Genomes ◽

Amplification Method ◽

Metagenome Sequencing

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Single Stranded DNA Viruses Associated with Capybara Faeces Sampled in Brazil

Viruses ◽

10.3390/v11080710 ◽

2019 ◽

Vol 11 (8) ◽

pp. 710 ◽

Cited By ~ 10

Author(s):

Rafaela S. Fontenele ◽

Cristiano Lacorte ◽

Natalia S. Lamas ◽

Kara Schmidlin ◽

Arvind Varsani ◽

...

Keyword(s):

Urban Areas ◽

Viral Metagenomics ◽

Viral Diversity ◽

Limited Information ◽

Single Stranded Dna ◽

Dna Viruses ◽

Rural And Urban Areas ◽

Rural And Urban ◽

Hydrochoerus Hydrochaeris ◽

Distrito Federal

Capybaras (Hydrochoerus hydrochaeris), the world’s largest rodents, are distributed throughout South America. These wild herbivores are commonly found near water bodies and are well adapted to rural and urban areas. There is limited information on the viruses circulating through capybaras. This study aimed to expand the knowledge on the viral diversity associated with capybaras by sampling their faeces. Using a viral metagenomics approach, we identified diverse single-stranded DNA viruses in the capybara faeces sampled in the Distrito Federal, Brazil. A total of 148 complete genomes of viruses in the Microviridae family were identified. In addition, 14 genomoviruses (family Genomoviridae), a novel cyclovirus (family Circoviridae), and a smacovirus (family Smacoviridae) were identified. Also, 37 diverse viruses that cannot be assigned to known families and more broadly referred to as unclassified circular replication associated protein encoding single-stranded (CRESS) DNA viruses were identified. This study provides a snapshot of the viral diversity associated with capybaras that may be infectious to these animals or associated with their microbiota or diet.

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Discovery of Four Novel Circular Single-Stranded DNA Viruses in Fungus-Farming Termites

Genome Announcements ◽

10.1128/genomea.00318-18 ◽

2018 ◽

Vol 6 (17) ◽

Cited By ~ 5

Author(s):

Mason Kerr ◽

Karyna Rosario ◽

Christopher C. M. Baker ◽

Mya Breitbart

Keyword(s):

Termite Mounds ◽

Single Stranded Dna ◽

Dna Viruses ◽

Content Type ◽

The Family

ABSTRACT Here, we describe four novel circular single-stranded DNA viruses discovered in fungus-farming termites ( Odontotermes sp.). The viruses, named termite-associated circular virus 1 (TaCV-1) through TaCV-4, are most similar to members of the family Genomoviridae and were widely detected in African termite mounds.

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