The Effect of Preprocessing by Sequence-Independent, Single-Primer Amplification (SISPA) on Metagenomic Detection of Viruses

Karlsson, Oskar E.; Belák, Sándór; Granberg, Fredrik

doi:10.1089/bsp.2013.0008

Cited by 49 publications

(39 citation statements)

References 55 publications

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“…MDA preferentially amplifies circular ssDNA viruses leading to an overrepresentation of this group in the final datasets [49–51]. In contrast, RP-SISPA is biased towards the most abundant viruses, as well as those with the largest genomes, and will therefore underrepresent small or less abundant ssDNA viruses [25, 52]. Other technical factors in the extraction and analysis protocols will have introduced additional biases.…”

Section: Discussionmentioning

confidence: 99%

Environmental drivers of viral community composition in Antarctic soils identified by viromics

et al. 2017

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BackgroundThe Antarctic continent is considered the coldest and driest place on earth with simple ecosystems, devoid of higher plants. Soils in the ice-free regions of Antarctica are known to harbor a wide range of microorganisms from primary producers to grazers, yet their ecology and particularly the role of viruses is poorly understood. In this study, we examined the virus community structures of 14 soil samples from the Mackay Glacier region.MethodsViral communities were extracted from soil and the dsDNA was extracted, amplified using single-primer amplification, and sequenced using the Ion Torrent Proton platform. Metadata on soil physico-chemistry was collected from all sites. Both read and contig datasets were analyzed with reference-independent and reference-dependent methods to assess viral community structures and the influence of environmental parameters on their distribution.ResultsWe observed a high heterogeneity in virus signatures, independent of geographical proximity. Tailed bacteriophages were dominant in all samples, but the incidences of the affiliated families Siphoviridae and Myoviridae were inversely correlated, suggesting direct competition for hosts. Viruses of the families Phycodnaviridae and Mimiviridae were present at significant levels in high-diversity soil samples and were found to co-occur, implying little competition between them. Combinations of soil factors, including pH, calcium content, and site altitude, were found to be the main drivers of viral community structure.ConclusionsThe pattern of viral community structure with higher levels of diversity at lower altitude and pH, and co-occurring viral families, suggests that these cold desert soil viruses interact with each other, the host, and the environment in an intricate manner, playing a potentially crucial role in maintaining host diversity and functioning of the microbial ecosystem in the extreme environments of Antarctic soil.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0301-7) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Environmental drivers of viral community composition in Antarctic soils identified by viromics

et al. 2017

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“…We recommend the use of the RP-SISPA methods presented here for amplifying viral DNA and RNA isolated from coral tissue using mechanical disruption. RP-SISPA has been used previously to detect and identify viruses through a metagenomic approach (Culley et al, 2010; Karlsson et al, 2013), and has an advantage over MDA in that branching does not occur during amplification, the formation of chimeras is reduced and ssDNA is not preferentially amplified (Polson et al, 2011). However, RP-SISPA is not without its own issues and has been reported to show a template-dependent amplification bias, resulting in uneven sequencing depth within and among genomes (Victoria et al, 2009, 2010; Rosseel et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

“…However, RP-SISPA is not without its own issues and has been reported to show a template-dependent amplification bias, resulting in uneven sequencing depth within and among genomes (Victoria et al, 2009, 2010; Rosseel et al, 2013). RP-SISPA can have a bias for amplifying the dominant sequences in metagenomic samples (Karlsson et al, 2013), likely the reason that most of the sequences in the RP-SISPA metagenomes contain sequences from the dsDNA viruses, which have larger genomes relative to other viral types. In addition, PCR amplification is known to produce amplification artifacts, such as increased heteroduplex formation in samples with increased diversity (Qiu et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Generating viral metagenomes from the coral holobiont

et al. 2014

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† These authors have contributed equally to this work.Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available) the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

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“…Desert soils frequently produce suboptimal (Յ10 ng/l) viral DNA yields (66), forcing the inclusion of a random PCR amplification step for NGS library construction. The use of whole-genome amplification (WGA) by multiple-displacement amplification (MDA) or random-priming, sequence-independent, single-primer amplification (RP-SISPA) (67) almost certainly results in biased amplification of certain virus groups (68)(69)(70)(71) and prevents the accurate determination of viral abundances and diversity. While viral amplification is widely accepted as a necessity in metaviromic studies (72), we argue that amplification of virus metagenomic DNA should be avoided where possible.…”

Section: Technical Recommendations and Future Researchmentioning

confidence: 99%

Diversity and Ecology of Viruses in Hyperarid Desert Soils

Zablocki

Adriaenssens

Cowan

2016

Appl Environ Microbiol

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In recent years, remarkable progress has been made in the field of virus environmental ecology. In marine ecosystems, for example, viruses are now thought to play pivotal roles in the biogeochemical cycling of nutrients and to be mediators of microbial evolution through horizontal gene transfer. The diversity and ecology of viruses in soils are poorly understood, but evidence supports the view that the diversity and ecology of viruses in soils differ substantially from those in aquatic systems. Desert biomes cover ϳ33% of global land masses, and yet the diversity and roles of viruses in these dominant ecosystems remain poorly understood. There is evidence that hot hyperarid desert soils are characterized by high levels of bacterial lysogens and low extracellular virus counts. In contrast, cold desert soils contain high extracellular virus titers. We suggest that the prevalence of microbial biofilms in hyperarid soils, combined with extreme thermal regimens, exerts strong selection pressures on both temperate and virulent viruses. Many desert soil virus sequences show low values of identity to virus genomes in public databases, suggesting the existence of distinct and as-yet-uncharacterized soil phylogenetic lineages (e.g., cyanophages). We strongly advocate for amplification-free metavirome analyses while encouraging the classical isolation of phages from dominant and culturable microbial isolates in order to populate sequence databases. This review provides an overview of recent advances in the study of viruses in hyperarid soils and of the factors that contribute to viral abundance and diversity in hot and cold deserts and offers technical recommendations for future studies.

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The Effect of Preprocessing by Sequence-Independent, Single-Primer Amplification (SISPA) on Metagenomic Detection of Viruses

Cited by 49 publications

References 55 publications

Environmental drivers of viral community composition in Antarctic soils identified by viromics

Environmental drivers of viral community composition in Antarctic soils identified by viromics

Generating viral metagenomes from the coral holobiont

Diversity and Ecology of Viruses in Hyperarid Desert Soils

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