2015
DOI: 10.1038/srep16532
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Modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis

Abstract: A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This… Show more

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Cited by 287 publications
(310 citation statements)
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“…25 Fecal suspensions (10% w/v in universal transport medium) were homogenized for 1 min at 3000 rpm with a MINILYS homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) and filtered using a 0.8 μm PES filter (Sartorius, Goettingen, Germany). The filtrate was then treated with a cocktail of Benzonase (Millipore, Billerica, MA, USA) (Novagen, Madison, WI, USA) and Micrococcal Nuclease (New England Biolabs, Ipswich, MA, USA) at 37 °C for 2 h to digest free-floating nucleic acids.…”
Section: Methodsmentioning
confidence: 99%
“…25 Fecal suspensions (10% w/v in universal transport medium) were homogenized for 1 min at 3000 rpm with a MINILYS homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) and filtered using a 0.8 μm PES filter (Sartorius, Goettingen, Germany). The filtrate was then treated with a cocktail of Benzonase (Millipore, Billerica, MA, USA) (Novagen, Madison, WI, USA) and Micrococcal Nuclease (New England Biolabs, Ipswich, MA, USA) at 37 °C for 2 h to digest free-floating nucleic acids.…”
Section: Methodsmentioning
confidence: 99%
“…Each bat was held in paper sack for 5 to 20 minutes allowing enough time for fresh feces to be produced after which the bats were released. Then, 25 pools were made from 87 collected samples (85 from Eidolon helvum and 2 from Epomophorus gambianus) and the pools were treated to enrich for viral particles using the NetoVIR protocol [15]. Sequencing of the samples was performed on a HiSeq 2500 platform (Illumina) for 300 cycles (2x150 bp paired ends).…”
Section: Methodsmentioning
confidence: 99%
“…First, only a small fraction of the total nucleic acids are of known viral origin, hence mechanical and enzymatic viral purification is often needed [9]. Second, the low abundance of viral particles in the samples requires the use of viral concentration methods prior to nucleic acid extraction [11] and is often combined with subsequent random DNA amplification [12]. Third, the nucleic acid extraction procedure has to cover the large variety in viral structures and genome types.…”
Section: Introductionmentioning
confidence: 99%