Methods in Viral Metagenomics

Thurber, Rebecca Vega

doi:10.1002/9781118010549.ch3

Cited by 8 publications

(7 citation statements)

References 73 publications

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“…This result is consistent with findings from a recent hot desert hypolith metavirome sequence analysis (Adriaenssens et al ., ) and supports the conclusion that these members of the Caudovirales are widespread in hot deserts. Notably, the proportion of the three families ( Myoviridae , Siphoviridae and Podoviridae ) in the Caudovirales have only slightly different values from those reported by Adriaenssens and co‐workers (Adriaenssens et al ., ) (http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.13088/suppinfo), perhaps a surprising result given the known biases of the multiple displacement amplification protocol (Thurber, ) underlying this metaviromics study.…”

Section: Resultsmentioning

confidence: 99%

Metagenomic analysis provides insights into functional capacity in a hyperarid desert soil niche community

Vikram

Guerrero

Makhalanyane

et al. 2015

Environmental Microbiology

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Environmental stressors such as low water activity and temperature extremes impose severe limitations on the productivity of soils in hyperarid deserts. In such ecosystems, macroscopic communities are often restricted to cryptic niche habitats, such as hypoliths (microbial communities found beneath translucent rocks), which are widely distributed in hyperarid desert environments. While hypolithic communities are considered to play a major role in the productivity of hyperarid habitats, the functional guilds implicated in these processes remain unclear. Here, we describe the Illumina-based metagenomic sequencing (± 30 Gb), assembly and analysis of hypolithic microbial communities from the south-west African Namib Desert.Taxonomic analyses using Small Subunit (SSU) phylogenetic markers showed that bacterial phylotypes (93%) dominated the communities, with relatively small proportions of archaea (0.43%) and fungi (5.6%). BlastX analysis against the refseq-viral database showed the presence of double stranded DNA viruses (7.8% contigs), dominated by Caudovirales (59.2%). Analysis of functional genes and metabolic pathways revealed that cyanobacteria were primarily responsible for photosynthesis with the presence of multiple copies of genes for both photosystems I and II, with a smaller but significant fraction of proteobacterial anoxic photosystem II genes. Hypolithic community members demonstrated an extensive genetic capacity for the degradation of phosphonates and mineralization of organic sulfur.Our data suggest that Proteobacterial guilds may be more significant in desert niches than previously recognized, as they showed widespread genetic capacity for mediating key stages in all biogeochemical cycles. Surprisingly, we were unable to show the presence of genes representative of complete nitrogen cycles. The diversity of nif genes was low, and the metagenome showed no evidence of other key N-cycling genes. Taken together, our analyses suggest an extensive capacity for carbon, phosphate and sulphate cycling but only limited nitrogen biogeochemistry. 2

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Section: Resultsmentioning

confidence: 99%

Metagenomic analysis provides insights into functional capacity in a hyperarid desert soil niche community

Vikram

Guerrero

Makhalanyane

et al. 2015

Environmental Microbiology

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“…A broad range of methods has been described to purify and concentrate viral particles. These include tangential flow filtration (TFF) [ 40 , 44 , 45 ], PEG precipitation [ 18 , 19 , 46 ], cesium chloride (CsCl) gradient ultracentrifugation [ 22 , 32 , 44 ], and more recently iodixanol (Optiprep TM ) density gradient medium. The main disadvantage of CsCl ultracentrifugation is that when analyzing the viral communities of an environment, the lack of prior knowledge of the viruses constituting these communities prevents the harvesting of specific fractions where viruses are located, resulting in recovery of all fractions and thereby diluting the viruses.…”

Section: Discussionmentioning

confidence: 99%

“…Viral metagenomic analyses of complex environments usually require pre-treatment steps, such as viral purification and nucleic acid enrichment, before sequencing. Several physical characteristics of viral particles enable viral purification (e.g., capsid durability), but the wide variety of viruses’ biological characteristics cause difficulties in developing a standardized protocol compatible with a broad range of particle sizes, shapes, densities, and genome types [ 18 ]. Usually, virome preparation is based on dead-end or tangential flow filtration, and nuclease digestion of non-protected viral and host cells.…”

Section: Introductionmentioning

confidence: 99%

“…During the last decade, several standardized protocols for generating DNA viromes from various environments have been described [ 18 – 20 ], but such standardization has not been reached so far for RNA viruses. Here, we present a simple protocol for the purification and sequencing of RNA viromes from host-associated biological samples of various origins.…”

Section: Introductionmentioning

confidence: 99%

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Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes

et al. 2015

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BackgroundMetagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles.Principal FindingsWe evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7–15% of all reads were classified as “unknown”, depending on the random amplification method.ConclusionThe protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses.

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“…Epifluorescence microscopy was performed at every step to monitor the absence of bacterial contamination in the final concentrates and DNAse I reactions were performed to further ensure that VLP concentrates were free of any contamination with environmental DNA. For DNA isolation, formamide/CTAB extractions followed by phenol/chloroform purifications were performed (Thurber, 2011).…”

Section: Sample Processing and Dna Isolationmentioning

confidence: 99%

Analysis of viral and bacterial communities in groundwater associated with contaminated land

Costeira

Doherty

Allen

et al. 2019

Science of The Total Environment

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Bacteriophages are considered to be key entities of various environments  Groundwater microbial communities were studied using molecular biology approaches  Phage and bacterial diversities were correlated with contamination and pH  Viruses of degraders were identified and phage-bacterial associations described  A total environmental community approach provides valuable insights towards bioremediation 2

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Methods in Viral Metagenomics

Cited by 8 publications

References 73 publications

Metagenomic analysis provides insights into functional capacity in a hyperarid desert soil niche community

Metagenomic analysis provides insights into functional capacity in a hyperarid desert soil niche community

Host-Associated Metagenomics: A Guide to Generating Infectious RNA Viromes

Analysis of viral and bacterial communities in groundwater associated with contaminated land

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