Novel Virus Identification through Metagenomics: A Systematic Review

Abstract

Metagenomic Next Generation Sequencing (mNGS) allows the evaluation of complex microbial communities, avoiding isolation and cultivation of each microbial species, and does not require prior knowledge of the microbial sequences present in the sample. Applications of mNGS include virome characterization, new virus discovery and full-length viral genome reconstruction, either from virus preparations enriched in culture or directly from clinical and environmental specimens. Here, we systematically reviewed studies that describe novel virus identification through mNGS from samples of different origin (plant, animal and environment). Without imposing time limits to the search, 379 publications were identified that met the search parameters. Sample types, geographical origin, enrichment and nucleic acid extraction methods, sequencing platforms, bioinformatic analytical steps and identified viral families were described. The review highlights mNGS as a feasible method for novel virus discovery from samples of different origins, describes which kind of heterogeneous experimental and analytical protocols are currently used and provides useful information such as the different commercial kits used for the purification of nucleic acids and bioinformatics analytical pipelines.

Keywords: NGS; animal; environment; novel virus; plant; viral metagenomics.

Figure 1

Flowchart of the literature search. Search was performed following the guidelines of the preferred reporting items for systematic reviews and meta-analyses (PRISMA).

Figure 2

Summary of the publications included in the study per year and per type of sample. (a) Trend for annual publication: each column represents the number of articles by year of publication, from 2008 to 2021, divided by type of sample (environment, animal, plant). (b) Percentage of each sample type of the 379 publications: animal (dark blue), environment (light blue), yellow (plant).

Figure 3

Sample size and geographical provenience. (a) Number of publications for each sample size range; X axes: number of publications, Y axes; sample size range. ND: sample size not specified. (b) Map showing the geographical provenance of the samples; dots indicate the samples/publications (dot dimension reflects different publication numbers). (b) was generated with Bing Maps add-on for Microsoft Office Excel.

Figure 4

Sample enrichment and purification. (a) Enrichment methods used for environmental samples prior to extraction. The figure shows the different enrichment methods and the percentage of each method of the total number of publications (environmental sample). (b) Extraction method. The graph shows the different extraction methods used for all samples. X axis: publication number; Y axis: extraction method.

Figure 5

Sequencing platforms. (a) The number of publications employing the different platforms. (b) Annual publications for each sequencing platform. Each column represents the number of articles by year of publication, from 2008 to 2021, divided by platform. “Published sequences” refers to analysis of previously published sequences.

Figure 6

An overview of 51 included publications (year 2021). Each column represents one paper, and numbers refer to the list of references provided in Supplementary File S1. Each row describes one specific characteristic (from top to bottom): sample, enrichment method, sample size, viral genome, sequencing platform, de novo assembly, retro-transcription (RT), provenance of the sample (WHO classification) and extraction method. Categories and color codes for each data field are indicated in the figure legend. Gray boxes indicate that the technique was not performed or the information was not provided.

Figure 7

Viral families discovered in different sample types. The figure shows the viral families and the number of new viruses belonging to each family in (a) animal samples; “other”: families that have been described in less than eight reports; ND: viruses not belonging to known families; (b) environmental samples; “other”: families that have been described in a single report; ND: viruses not belonging to known families; (c) plant samples; “other”: families that have been described in a single report. For all sample types, “unclassified”: as defined in NCBI taxonomy and viral zone databases.

Figure 8

Viral Genomes. The graph shows viral genomes of the identified viruses: DNA (blue bar), RNA (orange bar) and unclassified (gray bar) identified in the three sample types (animal, environment, plant). The number of viruses (percentage and absolute number) for each genome type is indicated on the side of the bars. X axis: DNA or RNA genomes in different sample types; y-axis: viral genomes.