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Viral Metagenomics

Overview

What Is Viral Metagenomics

A virus is a non-cellular organism of microscopic dimensions and simple structure, that contains a single type of nucleic acid, either DNA or RNA. In order to proliferate, viruses necessitate a parasitic existence within living cells, appropriating their host's machinery for replication. Viruses stand as the most abundant "biological" entities on Earth, pervading all known ecosystems—from aquatic environments and soil matrices to the human body, their ubiquity is unchallenged. Additionally, the capacity of viruses to infect all known types of organisms is universally recognized, encompassing fauna, flora, and microbes, inclusive of bacteria and archaea.

Viral Metagenomics, a burgeoning discipline anchored in the principles of metagenomics and bolstered by contemporary virus molecular biology detection methods, takes an all-inclusive approach to viral research. It directly targets all viral genetic material present in a sample— an assemblage typically referred to as the "Virome"— offering a swift and precise identification of all constituent viruses within the sample. This branch of science boasts diverse applicability, extending its reach to human or animal gut or blood samples, water and soil samples, to name a few. Not only does it play a pivotal role in various areas of research such as virus discovery, virus tracing, and microbial warning, but it also holds great significance in exploring the origins and evolution patterns of viruses, their genetic diversity, and geographical distribution.

Research into viral metagenomics commenced in 2002 when Bretbart and colleagues utilized metagenomic technologies to study two uncultivated marine viral communities, identifying bacteriophages as the main viral type in seawater. Recently, viral metagenomics research has undergone explosive growth as the role of viruses in regulating microbial ecological balance is increasingly noted. However, the number of known virus types is limited to a little over 6000 due to traditional virus cultivation methods' constraints. The development and widespread application of high-throughput sequencing technologies lead to the discovery of increasing numbers of unknown viruses.

Our Advantages:

Nearly all known and novel viruses in the sample can be analyzed using high-throughput sequencing and random PCR.
Culture-independent. The viruses are obtained directly from the environment.
We can systematically analyze and identify viruses which are too scattered or rare in the sample.
We provide comprehensive information on the biodiversity, taxonomy, and function of viruses in the sample.
We ensure strict quality control systems to guarantee accuracy.
Extensive experience and professional analysis.

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Applications of Viral Metagenomics

The application of viral metagenomics spans a broad array of domains, including agriculture, medicine, and ecology. In agriculture, viral metagenomics can be employed for extensive detection and monitoring of potential viruses in crops, enabling farmers to implement preventative and control measures promptly. Furthermore, understanding the evolutionary trajectories of viral strains and their relation to pathogenicity aids in the development of more effective disease-resistant varieties and vaccines, thereby enhancing the disease resistance of crops.

Medical science benefits from the holistic insights into the characteristics and variations of different viral strains that viral metagenomics provides. These insights facilitate epidemiological research on viruses, and vaccine design. For the discovery and classification of novel viruses, particularly those difficult to identify due to high genetic diversity, viral metagenomics offers a powerful tool. It also paves the way for a deeper understanding of virus-host interactions, uncovering molecular mechanisms during viral infection, and thus offering new targets and strategies for addressing viral diseases.

The impact of viruses on biodiversity is a pivotal component of ecosystem stability. In the field of ecology, viral metagenomics assists researchers in investigating the effects of virus-host interactions on biodiversity.

Service Specifications

Introduction to Our Viral Metagenomics Service

Viral metagenomics, the application of environmental genomics in the realm of viruses, necessitates the synthesis of innovative technologies such as virus manipulation, random PCR, Next-Generation Sequencing (NGS) and long-read sequencing. Techniques employed include sample preparation, virus particle enrichment, DNA library construction, sequencing and bioinformatics analysis. Illumina HiSeq/MiSeq, PacBio RS II, and Nanopore SMRT are amongst the common sequencing platforms. The efficiency of both viral metagenomics and bioinformatics analysis has vastly improved in recent years, with the former surpassing traditional viral research in detecting viruses of low abundance.

Viral metagenomic studies contribute to our understanding of viral ecology by shedding light on the genetic potential, community structure, and biogeography of environmental viruses. This field expands present knowledge on virus-host interactions by uncovering genes potentially enabling viruses to manipulate hosts in unexpected ways. The internal potential of viruses unveiled through viral metagenomics can aid in propelling a wide range of disciplines inclusive of evolutionary biology, pathogen monitoring and biotechnology. Moreover, functional viral metagenomics can serve as a platform for the discovery of more useful enzymes. As the annotation of virus genes continues to be perfected, the discovery of vital enzymes will markedly increase.

Technical Platforms

Illumina platforms
PE150
>1G clean data

Bioinformatics Analysis

Our bioinformatics analysis includes five parts: quality control, genomic assembly, taxonomic assignment, diversity analysis, functional analysis, and custom analysis. For advanced bioinformatics analysis in detail, please refer to the following table.

Bioinformatics Analysis	Details
Quality control	Fastqc, cutadapt, fastp, etc.
Genome assembly	Genovo, Bambus 2, Spades, MetaVelvet, Meta-IDBA, MEGAHIT, Ray, et al.
Taxonomic assignment	NCBI nr/nt database; Bowtie, BWA, SOAP3, mrsFAST, et al.
Diversity analysis	PcoA, NMDS, Heatmap, Krona analysis, clustering tree, etc.
Functional analysis	ARDB, CAZY, KEGG, eggNOG, GO, MG-RAST, IMG/M, WebMGA, RAMMCAP, etc.
Custom analysis	RDA/CCA, correlation analysis, ROC curve, etc.

Viral Metagenomics workflow

viral-metagenomics-2-1

Sample Requirement

DNA amount ≥ 3 μg, concentration ≥ 30 ng/μL, 1.8 < OD260/280 < 2.0.
Non-degraded DNA is placed into DNase-free tubes with clear labels. Please use dry ice during transport.
Sampling kits: We provide a complete range of microbial sampling kits for clients, including microbial collection products, DNA/RNA isolation kits, and accessories for storage and mailing.

Deliverables

Raw sequencing data (FASTQ)
Quality-control dashboard
Statistic data
Your designated bioinformatics reports

FAQs

Viral Metagenomics FAQs

Can metagenomics detect RNA viruses?
- Metagenomic techniques can be utilized for the detection of RNA viruses. Within the field of metagenomics, it is feasible to perform concurrent sequencing and analysis of substantial quantities of both DNA and RNA located within samples. As pertains to RNA viruses, the virus-specific RNA can be transcribed into cognate DNA prior to sequencing. This methodology permits the identification of a variety of RNA viruses present in the sample, inclusive of single-stranded and double-stranded RNA viruses, among others.
What is the use of metagenomics to describe viral diversity?
- Viral metagenomics can unveil the strain-based diversity intrinsic to virology. Harnessing metagenomic techniques fosters a comprehensive sequencing and incisive analysis of virus populations, shedding light on the sequence variation and differentiation between distinct viral strains. Such understanding is instrumental in deciphering the taxonomy and evolutionary relationships of viral strains, thereby augmenting accuracy within the domain of virus classification. Furthermore, metagenomics is adept at detecting an array of unknown or novel viral sequences encapsulated within samples. Analyzing these sequences paves the way for the identification of innovative virus types, invariably enhancing our understanding of viral diversity.
What are the challenges in the analysis of viral metagenomes?
- One of the main challenges of viral metagenomics stems from the diverse nature of viral nucleic acid types. Viruses, for example, can be categorized into double-stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, and single-stranded RNA viruses, etc. In addition, due to RNA viruses mutating at a faster pace than DNA viruses—coupled with the fact that DNA is easier to process from samples while RNA is unstable and requires more steps for RNA metagenomic analysis (reverse transcription)—there is an observable bias for DNA viruses in reference databases.
  
  Finally, the issue of sequence contamination from host organisms is significant. The source of the sample and preprocessing methodology can likely affect subsequent analysis results. Therefore, rigorous quality control measures and sample processing procedures need to be applied.
What is the difference between 16S rRNA and metagenomics?
- The use of metagenomics enables researchers to comprehensively study all types of microbes within a given community by providing exhaustive genomic information. Yet, despite offering high levels of precision and resolution, metagenomics involves complex data processing and analysis. Its inherent characteristics, however, make it highly efficient in meticulously identifying microbial species, and delving into their functions and metabolic potential.
  
  On the contrary, 16S rRNA sequencing allows for limited insights into 16S rRNA genes within the microbial community. With its comparatively reduced resolution, 16S rRNA sequencing appears simpler with regards to data treatment and analysis, making it a frequent choice for exploring phylogenetic relationships among microbes.
  
  More detailed descriptions of the differences are given in Microbial Species Identification Methods: 16S rRNA and Metagenomic Sequencing.

Demo

viral-metagenomics-3-1

Intestinal virome in patients with alcoholic hepatitis. (Jiang et al., 2020)

Multi-omics analyses show disease, diet, and transcriptome interactions with the virome. (Mihindukulasuriya et al., 2021)

Case Study

Viral metagenomics reveals diverse viruses in the fecal samples of children with diarrhea
Journal: Virologica Sinica
Impact factor: 5.573
Published: February 2022

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Backgrounds

In less industrialized nations, particularly Southeast Asia and sub-Saharan Africa, diarrheal diseases represent the third leading cause of mortality in children under five years of age. While recent improvements in sanitation and living standards have notably reduced the incidence and mortality rate of diarrheal diseases, childhood diarrhea resultant from viral infections remains a significant public health concern in developing countries. Viruses are the primary pathogens in such cases, accounting for 75% of total incidences. Array of viruses causing diarrhea includes Human Adenovirus (HAdV), Norovirus (NoV), Sapovirus (SaV), Human Astrovirus (HAstV), Rotavirus, Enterovirus, and Picornaviruses. However, knowledge about the prevalence of these viruses in children suffering from diarrhea remains considerably limited to date.

Methods

Sample preparation:

One hundred fresh stool samples
Viral DNA and RNA extraction

Viral Metagenomics Sequencing:

Reverse transcription synthesizes
Library construction
Miseq Illumina platform

Data analyses

Bioinformatics analysis
Phylogenetic analysis

Results

1. Viral metagenomic overview

In this study, analysis of 100 fecal samples from children with diarrhea revealed that among the eukaryotic viral families, Caliciviridae predominated at 78.42%, followed by Adenoviridae (8.94%), Picornaviridae (8.36%), Reoviridae (2.81%), Astroviridae (1.18%), Reoviridae (0.20%), Anelloviridae (0.04%), and Picobirnaviridae (0.03%).

Fig. 1. Taxonomic analysis of fecal virome detected in diarrheal children on the family level. A The composition of fecal virome detected in diarrheal children. The percentage of virus sequences in different virus family was shown. B Heatmap representing the reads number of each viral family in exponential form.

2. Phylogenetic analysis

In this study, nine viruses belonging to two genera of the Caliciviridae family were identified, of which eight were categorized as Norovirus (NoV), and one was part of the Sapovirus (SaV) genus. Seven viruses were under two genera of Picornaviridae, with three nearly full Enterovirus (EV) genomes and four almost complete sequences of Human parechoviruses. In addition, the presence of viruses belonging to the Mamastrovirus, Bocavirus, Mastadenovirus, and Rotavirus genera was also reported. This analysis expands the current knowledge of virodiversity in cases of paediatric diarrhoea.

Fig. 2. The phylogenetic analysis based on the complete genome sequences of NoVs and SaVs.

Fig. 3. The phylogenetic analysis of picornaviruses identified in this study.

Fig. 4. The phylogenetic analysis of human rotaviruses identified in this study.

Conclusion

In this investigation, the researchers utilized viral metagenomic sequencing to analyze the viral communities present in fecal samples from children suffering from diarrhea. They observed that a significant proportion of DNA or RNA viruses associated with diarrhea belonged to six families: Adenoviridae, Astroviridae, Caliciviridae, Picornaviridae, Reoviridae, and Picobirnaviridae. Notably, Caliciviridae exhibited the highest prevalence at 78.42%, followed by Adenoviridae (8.94%) and Picornaviridae (8.36%). In addition to known pathogens implicated in human diarrheal diseases, the researchers identified unrelated viruses such as Circoviridae and Picobirnaviridae. This study advances our comprehension of the virome in pediatric diarrhea, furnishing valuable insights for the prevention and management of viral gastroenteritis.

References

Braga L P P, Coutinho F H, Amgarten D E, et al. Novel virocell metabolic potential revealed in agricultural soils by virus‐enriched soil metagenome analysis. Environmental microbiology reports, 2021, 13(3): 348-354.
Bai G H, Lin S C, Hsu Y H, et al. The human virome: viral metagenomics, relations with human diseases, and therapeutic applications. Viruses, 2022, 14(2): 278.
Coutinho F H, Gregoracci G B, Walter J M, et al. Metagenomics sheds light on the ecology of marine microbes and their viruses. Trends in microbiology, 2018, 26(11): 955-965.
Greninger A L. A decade of RNA virus metagenomics is (not) enough. Virus research, 2018, 244: 218-229.
Jiang L, Lang S, Duan Y, et al. Intestinal virome in patients with alcoholic hepatitis. Hepatology, 2020, 72(6): 2182-2196.
Mihindukulasuriya K A, Mars R A T, Johnson A J, et al. Multi-omics analyses show disease, diet, and transcriptome interactions with the virome. Gastroenterology, 2021, 161(4): 1194-1207. e8.
Shixing Yang, Yumin He, Ju Zhang, et al. Viral metagenomics reveals diverse viruses in the fecal samples of children with diarrhea. VIROLOGICA SINICA, 2022, 37(1): 82-93