Detecting contamination in viromes using ViromeQC

Zolfo, Moreno; Pinto, Federica; Asnicar, Francesco; Manghi, Paolo; Tett, Adrian; Bushman, Frederic D.; Segata, Nicola

doi:10.1038/s41587-019-0334-5

Cited by 83 publications

(100 citation statements)

References 32 publications

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“…In virome generation, virion purification techniques are incorporated to remove non-encapsidated DNA (e.g., DNase treatment to remove eDNA), but nevertheless non-encapsidated DNA is still detected [183,184]. One reason for this is that eDNA, including vleDNA, can be bound to inorganic or organic compounds that can prevent its degradation.…”

Section: Removing Edna From Environmental Samplesmentioning

confidence: 99%

“…Nevertheless, PMA treatment is still useful, as one environmental metagenomic study, in which samples were collected from a clean-room floor, found that removal of relic DNA allowed detection of microbes and viruses that were not otherwise detected due to their low prevalence relative to that of relic DNA [155]. Once PMA treatment is performed, or any method of eDNA removal, qPCR can be used with 16S or 18S rRNA gene primers [184] to check to see if microbial DNA is still present in a virome, either because microbial relic DNA was not removed during PMA treatment or microbial DNA remained within intact ultrasmall microbial cells (Section 4.3).…”

Section: Removing Edna From Environmental Samplesmentioning

confidence: 99%

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Coming-of-Age Characterization of Soil Viruses: A User’s Guide to Virus Isolation, Detection within Metagenomes, and Viromics

et al. 2020

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The study of soil viruses, though not new, has languished relative to the study of marine viruses. This is particularly due to challenges associated with separating virions from harboring soils. Generally, three approaches to analyzing soil viruses have been employed: (1) Isolation, to characterize virus genotypes and phenotypes, the primary method used prior to the start of the 21st century. (2) Metagenomics, which has revealed a vast diversity of viruses while also allowing insights into viral community ecology, although with limitations due to DNA from cellular organisms obscuring viral DNA. (3) Viromics (targeted metagenomics of virus-like-particles), which has provided a more focused development of ‘virus-sequence-to-ecology’ pipelines, a result of separation of presumptive virions from cellular organisms prior to DNA extraction. This separation permits greater sequencing emphasis on virus DNA and thereby more targeted molecular and ecological characterization of viruses. Employing viromics to characterize soil systems presents new challenges, however. Ones that only recently are being addressed. Here we provide a guide to implementing these three approaches to studying environmental viruses, highlighting benefits, difficulties, and potential contamination, all toward fostering greater focus on viruses in the study of soil ecology.

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Section: Removing Edna From Environmental Samplesmentioning

confidence: 99%

Section: Removing Edna From Environmental Samplesmentioning

confidence: 99%

Coming-of-Age Characterization of Soil Viruses: A User’s Guide to Virus Isolation, Detection within Metagenomes, and Viromics

et al. 2020

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“…While a higher abundance of SSaDV could reflect important host-virus biology, it remains to be determined whether greater viral loads, determined by qPCR, of SSaDV has any biological significance. A pitfall of viral metagenomics from animal tissue using a viral enrichment method is the significant variability in non-viral genetic material between viromes within a study making quantitative comparisons difficult (33). According to our metagenomic survey, SSaDV is one species within a diverse extant population of densovirus present in sea star populations on the west coast of the United States.…”

Section: Discussion Sectionmentioning

confidence: 99%

Diversity of sea star-associated densoviruses and transcribed endogenized viral elements of densovirus origin

Jackson

Wilhelm

Johnson

et al. 2020

Preprint

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A viral etiology of Sea Star Wasting Syndrome (SSWS) has been largely explored using metagenomics leading to the conclusion that a densovirus is the predominant DNA virus associated with this syndrome, and, thus, the most promising viral candidate pathogen. Single-stranded DNA viruses are however highly diverse and pervasive among eukaryotic organisms which we hypothesize may confound the association between densoviruses and SSWS in sea stars. To test this hypothesis and assess the association of densoviruses to SSWS, we compiled past metagenomic data with new metagenomic-derived viral genomes from sea stars collected from Antarctica, California, Washington, and Alaska. We used 179 publicly available sea star transcriptomes to complement our approaches for densovirus discovery. Lastly, we focus the study to SSaDV, the first sea star densovirus discovered, by documenting its biogeography and putative tissue tropism. Transcriptomes contained mostly endogenized densovirus elements similar to the NS1 gene, while >30 complete and near-complete densoviral genomes were recovered from viral metagenomes. SSaDV was associated with nearly all tested species from southern California to Alaska, and in contrast to previous work, we show SSaDV is one genotype among a high diversity of densoviruses present in sea stars across the west coast of the United States and globally that are commonly associated with grossly normal (i.e. healthy or asymptomatic) animals. The diversity and ubiquity of these viruses in wild sea stars confounds the original hypothesis that one densovirus was the etiologic agent of SSWD.ImportanceThe primary interest in sea star densoviruses, specifically SSaDV, has been their association with Sea Star Wasting Syndrome (SSWS), a disease that has decimated sea star populations across the west coast of the United States since 2013. The association of SSaDV to SSWS was originally drawn from metagenomic analyses concluding that it was (1) the only densovirus present in the metagenomic data and (2) the most likely viral candidate based on representation in symptomatic sea stars. We reassessed the original metagenomic data with additional genomic datasets and found that SSaDV was one of ten densoviruses present in the original dataset and was no more represented in symptomatic sea stars than in asymptomatic sea stars. Instead, SSaDV appears to be a widespread, generalist virus that exists among a large diversity of densoviruses present in sea star populations.

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“…Benchmark investigations have revealed that different viral enrichment protocols could generate different biases on the final virome product, mostly related with microbial contamination and biases against specific viruses (15)(16)(17)(18)(19). These findings call for strong caution on profiling and detection of VLPs in viromes, specifically when associations between pathologies and samples/microorganisms are claimed (20).…”

Section: Introductionmentioning

confidence: 99%

“…Presence of microbial genetic material has been broadly confirmed in a recent meta-analysis of viromes studies from human, animal and environmental samples. This highlighted how enrichment protocols can hinder the correct analyses of viral communities because most of the viromes were contaminated by bacterial, archaeal and fungal genetic material (18).…”

mentioning

confidence: 99%

A step-by-step sequence-based analysis of virome enrichment protocol for freshwater and sediment samples

Pinto

Zolfo

Beghini

et al. 2020

Preprint

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Cultivation-free metagenomic analysis afforded unprecedented details on the diversity, structure and potential functions of microbial communities in different environments. When employed to study the viral fraction of the community that is recalcitrant to cultivation, metagenomics can shed light into the diversity of viruses and their role in natural ecosystems. However, despite the increasing interest in virome metagenomics, methodological issues still hinder the proper interpretation and comparison of results across studies. Virome enrichment experimental protocols are key multi-step processes needed for separating and concentrating the viral fraction from the whole microbial community prior to sequencing. However, there is little information on their efficiency and their potential biases. To fill this gap, we used metagenomic and amplicon sequencing to examine the microbial community composition through the serial filtration and concentration steps commonly used to produce viral-enriched metagenomes. The analyses were performed on water and sediment samples from an Alpine lake. We found that, although the diversity of the retained microbial communities declined progressively during the serial filtration, the final viral fraction contained a large proportion (from 10% to 40%) of non-viral taxa, and that the efficacy of filtration showed biases based on taxonomy. Our results quantified the amount of bacterial genetic material in viromes and highlighted the influence of sample type on the enrichment efficacy. Moreover, since viral-enriched samples contained a significant portion of microbial taxa, computational sequence analysis should account for such biases in the downstream interpretation pipeline.ImportanceFiltration is a commonly used method to enrich viral particles in environmental samples. However, there is little information on its efficiency and potential biases on the final result. Using a sequence-based analysis on water and sediment samples, we found that filtration efficacy is dependent on sample type and that the final virome contained a large proportion of non-viral taxa. Our finding stressed the importance of downstream analysis to avoid biased interpretation of data.

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Detecting contamination in viromes using ViromeQC

Cited by 83 publications

References 32 publications

Coming-of-Age Characterization of Soil Viruses: A User’s Guide to Virus Isolation, Detection within Metagenomes, and Viromics

Coming-of-Age Characterization of Soil Viruses: A User’s Guide to Virus Isolation, Detection within Metagenomes, and Viromics

Diversity of sea star-associated densoviruses and transcribed endogenized viral elements of densovirus origin

A step-by-step sequence-based analysis of virome enrichment protocol for freshwater and sediment samples

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