Viral Metagenomic Sequencing

Bookmark and Share

As an experienced provider of NGS services and a partner of Illumina, CD Genomics is committed to offering unprecedented amounts of sequence data permitting rapid and innovative analytical approaches with cost-effective solutions. We have strong expertise in providing the confident and unbiased viral metagenomic sequencing service by employing state-of-the-art high throughput sequencers, sequencing strategies, and bioinformatics pipelines.

The Introduction of Viral Metagenomic Sequencing

Viruses are the most abundant biological entities in the world which outnumber microbial cells 10:1 in most environments. Viruses constantly inhabit our body, and asymptomatic hosts are no exceptions. This emerging vision raises the need for the exploration of the virome. Viral metagenomics can provide insights into the composition and structure of viral communities. Profiling the taxonomic composition of viral communities is important not only for basic research but also for clinical science and practice.

However, viral communities are difficult to be characterized because there is no single gene that is shared by all viral genomes, which limits the application of analogous methods used in bacteria for ribosomal DNA profiling. The low-abundance of free and cellular DNA is another challenge. Metagenomic shotgun sequencing and RNA-seq are two approaches for the characterization of viral communities. Direct sequencing can result in a high background of genetic materials. Therefore, extra procedures are needed to concentrate and purify viral particles (VPs). Multiple viable enrichment methods have been suggested, including filtration, ultracentrifugation, nuclease treatment, multiple displacement amplification (MDA), linker-amplified shotgun library (LASL), and random PCR approach.

Advantages of Viral Metagenomic Sequencing

  • No need for cultivation or antibody laboratory tests
  • Comprehensive information on community biodiversity, taxonomy, and function
  • Promising applications include virus discovery and viral pathogen identification associated with horticulture, veterinary medicine, and human health, especially for difficult-to-diagnose cases.

Viral Metagenomic Sequencing Workflow

Our highly experienced expert team executes quality management by following every procedure to ensure comprehensive and accurate results. The general workflow for viral metagenomic shotgun sequencing is outlined below. Briefly, the basic steps involved in viral metagenomics include the isolation and purification of viral particles by size filtration or density-based centrifugation, sequence-independent amplification of viral nucleic acid, shotgun sequencing or RNA-seq and matched analyses on diversity, taxonomy, and function through a battery of reliable bioinformatics tools.

Service Specifications

Sample requirements and preparation
  • Samples sources including environmental and clinical samples
  • Sample qualification control
  • Isolation and purification of VPs and sequence-independent amplification
Sequencing
  • HiSeq platforms, paired-end 150 bp, MGI DNBSEQ-T7/DNBSEQ-G400
  • More than 2Gb raw data per sample
  • More than 80% of bases with a ≥Q30 quality score
  • PacBio’s SMRT technology is also available for long fragment sequencing, which provides more accurate and contiguous sequences.
Bioinformatics Analysis
We provide multiple customized bioinformatics analyses:
  • The removal of host-related and bacteria-related contamination
  • Nucleotide alignment (VirFinder, VirusFinder and BLAST)
  • Protein alignment (RefSeq)
  • De novo assembly, taxonomy identification, coverage plot, and phylogenetic analysis
Analysis pipeline

Apart from the qualified viral metagenomic sequencing, we provide assistance, including the experimental design, the determination of the appropriate sequencing platform, software tools, and analysis methodologies, to accommodate your project. If you have additional requirements or questions, please do not hesitate to contact us, our experienced specialists would like to help you to solve your questions.

References:

  1. Li Y, et al. VIP: an integrated pipeline for metagenomics of virus identification and discovery. Scientific reports, 2016, 6: 3774
  2. Rampelli, S. et al. ViromeScan: a new tool for metagenomic viral community profiling. BMC Genomics. 2016, (17):165.
  3. Lewandowska D W, Zagordi O, Geissberger F D, et al. Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples. Microbiome, 2017, 5(1): 94.

1. What are the approaches to whole-genome sequencing of pathogens?

There are currently three main approaches to whole-genome sequencing of pathogens, i.e., metagenomic sequencing, PCR amplicon sequencing and target enrichment sequencing (Figure 1). Direct metagenomic sequencing provides an accurate and integrated representation of the sequences within a sample. PCR amplicon sequencing performs PCR reactions to enrich the viral genome, which substantially increases the workload for large genomes but reduces the costs. Target enrichment sequencing employs virus-specific nucleotide probes bound to a solid phase to enrich the viral genome in a single reaction, which decreases workload but increases the cost compared to PCR amplicon sequencing. The advantages and disadvantages are listed in Table 1. It shows that viral metagenomic sequencing has the unique advantages to investigate pathogen and characterize viral diversity in environmental and clinical samples.

Figure 1. The main approaches used for viral genome sequencing (Houldcroft et al. 2016).

Table 1. Advantages and disadvantages of different viral genome sequencing methods (Houldcroft et al. 2016).

Method Advantages Disadvantages
Metagenomics
  • Simple, cost-effective sample preparation
  • Sequence novel and poorly characterized genomes
  • Identify potential underlying pathogen
  • Low number of PCR cycles cause few biases and mutation
  • Reflects in vivo variation
  • Rapidness
  • High sequencing cost
  • Relatively low sensitivity to target pathogen
  • Large amounts of data analysis
  • Off-target pathogens may raise ethical and diagnostic issues
PCR amplicon
  • Reduced sequencing costs
  • Highly sensitive and well-covered even at a low pathogen load
  • PCR biases and mutation
  • Labour-intensive
  • Limited ability to identify novel pathogens
Target enrichment
  • Higher specificity
  • Overlapping probes increases tolerance for individual primer mismatches
  • Reflect in vivo variation
  • High cost and technical expertise for sample preparation
  • Unable to sequence novel pathogens
  • Low or incomplete coverage
  • Cost and time to generate probe sets

2、What is the general bioinformatics pipeline for viral metagenomic sequencing?

After sequencing, raw NGS reads are first preprocessed by the removal of adapter, low-quality, and low-complexity sequences, followed by computational subtraction of host-related reads. In a fast mode, viruses are identified by alignment to ViPR/IRD nucleotide DB. In a more sense mode, bacteria and related ribosomal RNA reads are removed before alignment to virus database. Unmatched reads are further aligned to a viral protein database (NCBI RefSeq DB). Then the taxonomy identification (Taxl), coverage plot (Covplot), de novo assembly (Multiple k-mer), and phylogenetic analysis (PhyGo) can be performed.

References:
1. Houldcroft C J, Beale M A, Breuer J. Clinical and biological insights from viral genome sequencing. Nature Reviews Microbiology, 2017, 15(3): 183-192.
2. Li Y, Wang H, Nie K, et al. VIP: an integrated pipeline for metagenomics of virus identification and discovery. Scientific reports, 2016, 6.

Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study

Journal: The Lancet Infectious Diseases
Impact factor: 19.864
Published: February 17, 2016

Background

The microcephaly cases in Brazil in 2015 were 20 times more than in previous years. Epidemiological data suggest that the incidence of microcephaly in Brazil might be associated with Zika virus. The authors took amniotic fluid samples from two pregnant women in Brazil whose fetuses were diagnosed with microcephaly, in efforts to investigate the cause of microcephaly.

Case Histories
  • Pregant women with symtoms of Zika virus infection
  • Fetuses diagosed with microcephaly
Sampling
  • 5mL of amniotic fluid samples from two women at gestational week 28
  • Serology tests
  • ELISA
Sequencing
  • Purification of viral particles
  • Quantitative reverse transcription PCR
  • Viral metagenomic sequencing
Analysis
  • PRINSEQ
  • BLAST
  • RefSeq
  • De novo assembly
  • Phylogenetic analysis

Results

1. Zika virus genome assembly

After removing cellular contamination, 288 904 sequences had similarity with virus genomes, and 683 sequences matched the Zika virus genome. Figure 1 shows the whole Zika virus genome isolated from patient 1 with gene annotation.

Figure 1. Comparative genome BLAST Atlas diagram of Zika virus. The green circle corresponds to the complete Brazilian Zika virus isolated from patient 1. The red circle corresponds to the Senegal strain of Zika virus. The blue circle corresponds to the Uganda strain.

2. Phylogenetic analysis

The phylogenetic analyses were done with the coding region for envelope (Figure 2) and NS5 genes (Figure 3), respectively. The geographical region of the Brazilian Zika virus strain could not be inferred due to sampling limitations, but it seemed more closely related to French Polynesia than African strains.

The geographical and chronological distributions of Zika virus lineages indicated that southeast Asian strain could have genetically isolated from African strains for approximately 50 years. This pattern was further confirmed by the genetic distance between the new Brazilian Zika virus sequences and the Ugandan Zika virus genome (Figure 4).

Figure 2. Maximum likelihood topologies of envelope genomic region from Brazilian Zika virus.

Figure 3. Maximum likelihood topologies of the NS5 genomic region from Brazilian Zika virus.

Figure 4. Maximum likelihood phylogeny of Brazilian Zika virus, other Flaviviridae genomes, and an alphavirus genome. DENV=dengue virus, JEV=Japanese encephalitis virus. YFV=yellow fever virus. ZIKA=Zika virus.

3. Zika virus infection could occur through the transplacental transmission

This article was the first to isolate the whole genome of Zika virus from amniotic fluid, which suggested that Zika virus infection could occur through the transplacental transmission.

References:

Calvet G, Aguiar R S, Melo A S O, et al. Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study. The Lancet infectious diseases, 2016, 16(6): 653-660.

For Research Use Only. Not for use in diagnostic procedures.

Featured Resources

Related Services

Quote Request

Contact CD Genomics

Terms & Conditions | Privacy Policy | Feedback   Copyright © CD Genomics. All rights reserved.
Top