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Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent.

ABSTRACT: Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries were compared for their abilities to detect multiple viruses and generate full genome coverage. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces.

SUBMITTER: Li L

PROVIDER: S-EPMC4344864 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent.

Li Linlin L Deng Xutao X Mee Edward T ET Collot-Teixeira Sophie S Anderson Rob R Schepelmann Silke S Minor Philip D PD Delwart Eric E

Journal of virological methods 20141211

Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries were compared for their abilities to detect multiple viruses and generate f ...[more]

PMID: 25497414

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Project description:The diversity and high genomic mutation rates of viral species hinder our understanding of viruses and their contributions to human health. Viral enterotypes as a description of the gut virome, its characteristics have not been thoroughly studied. Here we investigated the human gut virome composition using previously published sequencing data of 2,690 metagenomes from seven countries with various phenotypes. We found that the virome was dominated by double-stranded DNA viruses in our data, and young children and adults showed different stages in their fecal enterovirus composition. Beta diversity showed there were significantly less homogeneous in individuals with severe disorders of bile acid secretion, such as cirrhosis. In contrast, there were no significant differences in distances to centroids or viral components between patients with phenotypes unrelated to bile acid, such as hypertension. Enterotypes determined independently from various projects showed similar specific viruses and enrichment direction. Confounding factors, such as different sequencing platforms and library construction, did not confuse enterotyping. The gut virome composition pattern could be described by two viral enterotypes, which supported a discrete, rather than a gradient, distribution. Three main components, enterotype 1 and 2 specific viruses and the other, comprise the total viral variation in these sets. Compared with enterotype 2, enterotype 1 had a higher viral count, Shannon index, and similarity between samples. The relative abundance of enterotype-specific viruses is a crucial determinant of enterotype assignment. Samples not matching any of the defined enterotypes in the database did not necessarily correlate to sickness. Therefore, the background context must be carefully considered when using a viral enterotype as a feature for disease prediction. Our results highlight important insights into the human gut virome composition by exploring two-main viral enterotypes in population and providing an alternate covariate for early disease screening.

Dataset Information

Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent.

Publications

Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent.

Similar Datasets

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