Viral Metagenomics Research Articles

Nanopore and Illumina sequencing reveal different viral populations from human gut samples.

The advent of viral metagenomics, or viromics, has improved our knowledge and understanding of global viral diversity. High-throughput sequencing technologies enable explorations of the ecological roles, contributions to host metabolism, and the influence of viruses in various environments, including the human intestinal microbiome. However, bacterial metagenomic studies frequently have the advantage. The adoption of advanced technologies like long-read sequencing has the potential to be transformative in refining viromics and metagenomics. Here, we examined the effectiveness of long-read and hybrid sequencing by comparing Illumina short-read and Oxford Nanopore Technology (ONT) long-read sequencing technologies and different assembly strategies on recovering viral genomes from human faecal samples. Our findings showed that if a single sequencing technology is to be chosen for virome analysis, Illumina is preferable due to its superior ability to recover fully resolved viral genomes and minimise erroneous genomes. While ONT assemblies were effective in recovering viral diversity, the challenges related to input requirements and the necessity for amplification made it less ideal as a standalone solution. However, using a combined, hybrid approach enabled a more authentic representation of viral diversity to be obtained within samples.

Molecular characterization of plasma virome of hepatocellular carcinoma (HCC) patients

Hepatocellular carcinoma (HCC) stands as the most common cancer type, arising from various causes, and responsible for a substantial number of cancer-related fatalities. Recent advancements in viral metagenomics have empowered scientists to delve into the intricate diversity of the virosphere, viral evolution, interactions between viruses and their hosts, and the identification of viral causes behind disease outbreaks, the development of specific symptoms, and their potential role in altering the host’s physiology. The present study had the objective of “Molecular Characterization of HBV, HCV, anelloviruses, CMV, SENV-D, SENV-H, HEV, and HPV viruses among individuals suffering from HCC.” A total of 381 HCC patients contributed 10 cc of blood each for this study. The research encompassed the assessment of tumor markers, followed by molecular characterization of HBV, HCV, Anelloviruses (TTV, TTMV, and TTMDV), SENV-H and SENV-D viruses, HEV, CMV, and HPV, as well as histopathological examinations. The outcomes of this study revealed that majority of the HCC patients 72.4% (276/381) were male as compared to females. HCV infection, at 76.4% (291 out of 381), exhibited a significant association (p < 0.05) with HCC. Most patients displayed singular lesions in the liver, with Child Pugh Score Type B being the predominant finding in 45.2% of cases. Plasma virome analysis indicated the prevalence of TTMDV (75%), followed by TTMV (70%) and TTV (42.1%) among anelloviruses in HCC patients. Similarly, SENV-H (52%) was followed by SENV-D (20%), with co-infections at 15%. The presence of CMV and HEV among the HCC patients was recorded 5% each however 3.5% of the patients showed the presence of HPV. In conclusion, this study underscores that HCC patients serve as reservoirs for various pathogenic and non-pathogenic viruses, potentially contributing to the development, progression, and severity of the disease.

Comparison of Three Viral Nucleic Acid Preamplification Pipelines for Sewage Viral Metagenomics.

Viral metagenomics is a useful tool for detecting multiple human viruses in urban sewage. However, more refined protocols are required for its effective use in disease surveillance. In this study, we investigated the performance of three different preamplification pipelines (specific to RNA viruses, DNA viruses or both) for viral genome sequencing using spiked-in Phosphate Buffered Saline and sewage samples containing known concentrations of viruses. We found that compared to the pipeline targeting all genome types, the RNA pipeline performed better in detecting RNA viruses in both spiked and unspiked sewage samples, allowing the detection of various mammalian viruses including members from the Reoviridae, Picornaviridae, Astroviridae and Caliciviridae. However, the DNA-specific pipeline did not improve the detection of mammalian DNA viruses. We also measured viral recovery by quantitative reverse transcription polymerase chain reaction and assessed the impact of genetic background (non-viral genetic material) on viral coverage. Our results indicate that viral recoveries were generally lower in sewage (average of 11.0%) and higher in Phosphate Buffered Saline (average of 23.4%) for most viruses. Additionally, spiked-in viruses showed lower genome coverage in sewage, demonstrating the negative effect of genetic background on sequencing. Finally, correlation analysis revealed a relationship between virus concentration and genome normalized reads per million, indicating that viral metagenomic sequencing can be semiquantitative.

Snow viruses and their implications on red snow algal blooms.

Algal blooms can give snowmelt a red color, reducing snow albedo and creating a runaway effect that accelerates snow melting. The occurrence of red snow is predicted to grow in polar and subpolar regions with increasing global temperatures. We hypothesize that these algal blooms affect virus-bacteria interactions in snow, with potential effects on snowmelt dynamics. A genomic analysis of double-stranded DNA virus communities in red and white snow from the Whistler region of British Columbia, Canada, identified 792 putative viruses infecting bacteria. The most abundant putative snow viruses displayed low genomic similarity with known viruses. We recovered the complete circular genomes of nine putative viruses, two of which were classified as temperate. Putative snow viruses encoded genes involved in energy metabolisms, such as NAD+ synthesis and salvage pathways. In model phages, these genes facilitate increased viral particle production and lysis rates. The frequency of temperate phages was positively correlated with microbial abundance in the snow samples. These results suggest the increased frequency of temperate virus-bacteria interactions as microbial densities increase during snowmelt. We propose that this virus-bacteria dynamic may facilitate the red snow algae growth stimulated by bacteria.IMPORTANCEMicrobial communities in red snow algal blooms contribute to intensifying snowmelt rates. The role of viruses in snow during this environmental shift, however, has yet to be elucidated. Here, we characterize novel viruses extracted from snow viral metagenomes and define the functional capacities of snow viruses in both white and red snow. These results are contextualized using the composition and functions observed in the bacterial communities from the same snow samples. Together, these data demonstrate the energy metabolism performed by viruses and bacteria in a snow algal bloom, as well as expand the overall knowledge of viral genomes in extreme environments.

Two Novel Geminiviruses Identified in Bees (Apis mellifera and Nomia sp.).

Members of the Geminviridae family are circular single-stranded DNA plant-infecting viruses, some of which impact global food production. Geminiviruses are vectored by sap-feeding insects such as leafhoppers, treehoppers, aphids, and whiteflies. Additionally, geminivirus sequences have also been identified in other insects such as dragonflies, mosquitoes, and stingless bees. As part of a viral metagenomics study on honeybees and solitary bees (Nomia sp.), two geminivirus genomes were identified. These represent a novel citlodavirus (from honeybees collected from Westmoreland, Jamaica) and a mastrevirus-like genome (from a solitary bee collected from Tempe, Arizona, USA). The novel honeybee-derived citlodavirus genome shares ~61 to 69% genome-wide nucleotide pairwise identity with other citlodavirus genome sequences and is most closely related to the passion fruit chlorotic mottle virus identified in Brazil. Whereas the novel solitary bee-derived mastrevirus-like genome shares ~55 to 61% genome-wide nucleotide identity with other mastreviruses and is most closely related to tobacco yellow dwarf virus identified in Australia, based on pairwise identity scores of the full genome, replication-associated protein, and capsid protein sequences. Previously, two geminiviruses in the Begomovirus genus were identified in samples of stingless bee (Trigona spp.) samples. Here, we identify viruses that represent two new species of geminiviruses from a honeybee and a solitary bee, which continues to demonstrate that plant pollinators can be utilized for the identification of plant-infecting DNA viruses in ecosystems.

Metagenomic Analysis for Diagnosis of Hemorrhagic Fever in Minas Gerais, Brazil.

Viral hemorrhagic fever poses a significant public health challenge due to its severe clinical presentation and high mortality rate. The diagnostic process is hindered by similarity of symptoms across different diseases and the broad spectrum of pathogens that can cause hemorrhagic fever. In this study, we applied viral metagenomic analysis to 43 serum samples collected by the Public Health Laboratory (Fundação Ezequiel Dias, FUNED) in Minas Gerais State, Brazil, from patients diagnosed with hemorrhagic fever who had tested negative for the standard local hemorrhagic disease testing panel. This panel includes tests for Dengue virus (DENV) IgM, Zika virus IgM, Chikungunya virus IgM, yellow fever IgM, Hantavirus IgM, Rickettsia rickettsii IgM/IgG, and Leptospira interrogans IgM, in addition to respective molecular tests for these infectious agents. The samples were grouped into 18 pools according to geographic origin and analyzed through next-generation sequencing on the NextSeq 2000 platform. Bioinformatic analysis revealed a prevalent occurrence of commensal viruses across all pools, but, notably, a significant number of reads corresponding to the DENV serotype 2 were identified in one specific pool. Further verification via real-time PCR confirmed the presence of DENV-2 RNA in an index case involving an oncology patient with hemorrhagic fever who had initially tested negative for anti-DENV IgM antibodies, thereby excluding this sample from initial molecular testing. The complete DENV-2 genome isolated from this patient was taxonomically classified within the cosmopolitan genotype that was recently introduced into Brazil. These findings highlight the critical role of considering the patient's clinical condition when deciding upon the most appropriate testing procedures. Additionally, this study showcases the potential of viral metagenomics in pinpointing the viral agents behind hemorrhagic diseases. Future research is needed to assess the practicality of incorporating metagenomics into standard viral diagnostic protocols.

Characterization of the gut bacterial and viral microbiota in latent autoimmune diabetes in adults

Latent autoimmune diabetes in adults (LADA) is a heterogeneous disease characterized by autoantibodies against insulin producing pancreatic beta cells and initial lack of need for insulin treatment. The aim of the present study was to investigate if individuals with LADA have an altered gut microbiota relative to non-diabetic control subjects, individuals with type 1 diabetes (T1D), and individuals with type 2 diabetes (T2D). Bacterial community profiling was performed with primers targeting the variable region 4 of the 16S rRNA gene and sequenced. Amplicon sequence variants (ASVs) were generated with DADA2 and annotated to the SILVA database. The gut virome was sequenced, using a viral particle enrichment and metagenomics approach, assembled, and quantified to describe the composition of the viral community. Comparison of the bacterial alpha- and beta-diversity measures revealed that the gut bacteriome of individuals with LADA resembled that of individuals with T2D. Yet, specific genera were found to differ in abundance in individuals with LADA compared with T1D and T2D, indicating that LADA has unique taxonomical features. The virome composition reflected the stability of the most dominant order Caudovirales and the families Siphoviridae, Podoviridae, and Inoviridae, and the dominant family Microviridae. Further studies are needed to confirm these findings.

Isolation and Identification of Severe Fever with Thrombocytopenia Syndrome Virus from Farmed Mink in Shandong, China

Severe fever with thrombocytopenia syndrome (SFTS) virus, recently named Bandavirus dabieense, belongs to the genus Bandavirus of family Phenuiviridae, and it causes SFTS in humans with clinical symptoms including fever, thrombocytopenia, gastrointestinal symptoms, and leukocytopenia. However, there are few reports on the pathogenesis of SFTSV in animals. This study first isolated the SFTSV strain SD22-2 from sick-farmed mink. Viral metagenomics was used to detect SFTSV nucleotide in the clinical specimens obtained from symptomatic minks. Then, we isolated the virus using Vero and DH82 cells, and Real-Time Quantitative PCR (RT-qPCR), indirect immunofluorescence assay, transmission electron microscopy, and Western blotting identified it. Meanwhile, phylogenetic analysis based on partial L, M, and S segment sequences indicated that the mink-origin SFTSV strain SD22-2 belonged to genotype D and was genetically close to the HB2016-003 strain isolated from humans. Taken together, we isolated and identified an SFTSV from farmed mink that may be the reservoir hosts of SFTSV. We should pay more attention to farmed minks and biosecurity practices, and active surveillance at fur farms must be reviewed and enhanced.

Temporal Dynamics and Contribution of Phage Community to the Prevalence of Antibiotic Resistance Genes in a Full-Scale Sludge Anaerobic Digestion Plant.

Anaerobic digestion (AD) is an important biological resource recovery process, where microorganisms play key roles for material transformation. There has been some knowledge about the prokaryotic community and antibiotic resistance genes (ARGs) in AD, but there has been very limited knowledge of phages. In this study, samples from a full-scale AD plant were collected over 13 months, sequenced, and analyzed for viral and prokaryotic metagenomes. Totally, 3015 viral operational taxonomic units (vOTUs) were detected, mostly assigned to Caudoviricetes. The phage community had faster temporal variation than the prokaryotic community. Warm seasons harbored a higher abundance of both temperate phages and broad host-range phages. Seven ARGs of 6 subtypes were carried by 20 vOTUs, a representative ermT gene was synthesized and expressed, and the resistance activity in the host was examined, confirming the real activity of virus-carried ARGs in the AD process. Some of the ARGs were horizontally transferred between the phage and prokaryotic genomes. However, phage infection was not found to contribute to ARG transfer. This study provided an insight into the ecological patterns of the phage community, confirmed the antibiotic resistance activity of virus-carried ARGs, evaluated the contribution of phages on the ARG prevalence, and laid the foundation for the control strategies of the community and antibiotic resistance in the AD process.

Genomoviruses in Liver Samples of Molossus molossus Bats.

CRESS-DNA encompasses a broad spectrum of viruses documented across diverse organisms such as animals, plants, diatoms, fungi, and marine invertebrates. Despite this prevalence, the full extent of these viruses' impact on the environment and their respective hosts remains incompletely understood. Furthermore, an increasing number of viruses within this category lack detailed characterization. This investigation focuses on unveiling and characterizing viruses affiliated with the Genomoviridae family identified in liver samples from the bat Molossus molossus. Leveraging viral metagenomics, we identified seven sequences (MmGmV-PA) featuring a circular DNA genome housing two ORFs encoding replication-associated protein (Rep) and capsid protein (Cap). Predictions based on conserved domains typical of the Genomoviridae family were established. Phylogenetic analysis revealed the segregation of these sequences into two clades aligning with the genera Gemycirculavirus (MmGmV-06-PA and MmGmV-07-PA) and Gemykibivirus (MmGmV-01-PA, MmGmV-02-PA, MmGmV-03-PA, MmGmV-05-PA, and MmGmV-09-PA). At the species level, pairwise comparisons based on complete nucleotide sequences indicated the potential existence of three novel species. In summary, our study significantly contributes to an enhanced understanding of the diversity of Genomoviridae within bat samples, shedding light on previously undiscovered viral entities and their potential ecological implications.

Amplidiff: an optimized amplicon sequencing approach to estimating lineage abundances in viral metagenomes

BackgroundMetagenomic profiling algorithms commonly rely on genomic differences between lineages, strains, or species to infer the relative abundances of sequences present in a sample. This observation plays an important role in the analysis of diverse microbial communities, where targeted sequencing of 16S and 18S rRNA, both well-known hypervariable genomic regions, have led to insights into microbial diversity and the discovery of novel organisms. However, the variable nature of discriminatory regions can also act as a double-edged sword, as the sought-after variability can make it difficult to design primers for their amplification through PCR. Moreover, the most variable regions are not necessarily the most informative regions for the purpose of differentiation; one should focus on regions that maximize the number of lineages that can be distinguished.ResultsHere we present AmpliDiff, a computational tool that simultaneously finds highly discriminatory genomic regions in viral genomes of a single species, as well as primers allowing for the amplification of these regions. We show that regions and primers found by AmpliDiff can be used to accurately estimate relative abundances of SARS-CoV-2 lineages, for example in wastewater sequencing data. We obtain errors that are comparable with using whole genome information to estimate relative abundances. Furthermore, our results show that AmpliDiff is robust against incomplete input data and that primers designed by AmpliDiff also bind to genomes sampled months after the primers were selected.ConclusionsWith AmpliDiff we provide an effective, cost-efficient alternative to whole genome sequencing for estimating lineage abundances in viral metagenomes.

Co-infecting viruses of species Bovine rhinitis B virus (Picornaviridae) and Bovine nidovirus 1 (Tobaniviridae) identified for the first time from a post-mortem respiratory sample of a sheep (Ovis aries) in Hungary

In this study, a picornavirus and a nidovirus were identified from a single available nasopharyngeal swab (NPS) sample of a freshly deceased sheep, as the only vertebrate viruses found with viral metagenomics and next-generation sequencing methods. The sample was originated from a mixed feedlot farm in Hungary where sheep and cattle were held together but in separate stalls. Most of the sheep had respiratory signs (coughing and increased respiratory effort) at the time of sampling. Other NPS were not, but additional enteric samples were collected from sheep (n = 27) and cattle (n = 11) of the same farm at that time. The complete/nearly complete genomes of the identified viruses were determined using RT-PCR and Nanopore (MinION-Flonge) / Dye-terminator sequencing techniques. The results of detailed genomic and phylogenetic analyses indicate that the identified picornavirus most likely belongs to a type 4 genotype of species Bovine rhinitis B virus (BRBV-4, OR885914) of genus Aphthovirus, family Picornaviridae while the ovine nidovirus (OvNV, OR885915) – as a novel variant – could belong to the recently created Bovine nidovirus 1 (BoNV) species of genus Bostovirus, family Tobaniviridae. None of the identified viruses were detectable in the enteric samples using RT-PCR and generic screening primer pairs.Both viruses are well-known respiratory pathogens of cattle, but their presence was not demonstrated before in other animals, like sheep. Furthermore, neither BRBV-4 nor BoNVs were investigated in European cattle and/or sheep flocks, therefore it cannot be determined whether the presence of these viruses in sheep was a result of a single host species switch/spillover event or these viruses are circulating in not just cattle but sheep populations as well.Further studies required to investigate the spread of these viruses in Hungarian and European sheep and cattle populations and to identify their pathogenic potential in sheep.

Full annotation of viral metagenomics in different components from Chinese blood donors using next-generation sequencing.

Emerging viruses in the blood of healthy/qualified donors can seriously affect transfusion safety. However, the virus characteristics in different healthy blood donors and blood components are still not fully understood. Buffy coat (BC) and plasma specimens were collected from 32 whole blood donors, and platelet (PLT) and BC specimens from 30 apheresis platelet donors to explore the full annotation of viral metagenomics in different blood components from Chinese blood donors using next-generation sequencing technology. The study detected 56 viruses in the plasma and BC groups of whole blood donors. The plasma group had a significantly higher viral abundance and more types of viruses than the BC group. We detected 20 viruses in the PLT and BC groups of apheresis platelet donors. Viral abundance and types were significantly lower in the BC group than in the PLT group. According to β-diversity analysis, the plasma group had a significantly different community structure and composition than the BC group. Viral nucleic acid is found in the blood of healthy Chinese blood donors, with the highest concentration in plasma, which could explain the distribution of viruses in the blood of healthy individuals.

Virome characterization of diarrheic red-crowned crane (G. japonensis)

BackgroundThe red-crowned crane is one of the vulnerable bird species. Although the captive population has markedly increased over the last decade, infectious diseases can lead to the death of young red-crowned cranes while few virological studies have been conducted.MethodsUsing a viral metagenomics approach, we analyzed the virome of tissues of the dead captive red-crowned crane with diarrhea symptoms in Dongying Biosphere Reserve, Shandong Province, China and feces of individual birds breeding at the corresponding captive breeding center, which were pooled separately.ResultsThere is much more DNA and RNA viruses in the feces than that of the tissues. RNA virus belonging to the families Picornaviridae, and DNA viruses belonging to the families Parvoviridae, associated with enteric diseases were detected in the tissues and feces. Genomes of the picornavirus, genomovirus, and parvovirus identified in the study were fully characterized, which further suggested that infectious viruses of these families were possibly presented in the diseased red-crowned crane.ConclusionRNA virus belonging to the families Picornaviridae, and DNA viruses belonging to the families Genomoviridae and Parvoviridae were possibly the causative agent for diarrhea of red-crowned crane. This study has expanded our understanding of the virome of red-crowned crane and provides a baseline for elucidating the etiology for diarrhea of the birds.

The long and short of it: benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies.

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid assembly approaches that combine long- and short-read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock bacteriophage community of previously sequenced phage genomes and sequenced them using Illumina, Nanopore and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies 41 and 157 % higher than found in Illumina only assemblies, respectively. While the best PacBio assemblies had SNPs at frequencies 12 and 78 % higher than found in Illumina-only assemblies, respectively. Despite high-read coverage, long-read-only assemblies recovered a maximum of one complete genome from any assembly, unless reads were down-sampled prior to assembly. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short-read-only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes.

Eco-evolutionary dynamics of gut phageome in wild gibbons (Hoolock tianxing) with seasonal diet variations

It has been extensively studied that the gut microbiome provides animals flexibility to adapt to food variability. Yet, how gut phageome responds to diet variation of wild animals remains unexplored. Here, we analyze the eco-evolutionary dynamics of gut phageome in six wild gibbons (Hoolock tianxing) by collecting individually-resolved fresh fecal samples and parallel feeding behavior data for 15 consecutive months. Application of complementary viral and microbial metagenomics recovers 39,198 virulent and temperate phage genomes from the feces. Hierarchical cluster analyses show remarkable seasonal diet variations in gibbons. From high-fruit to high-leaf feeding period, the abundances of phage populations are seasonally fluctuated, especially driven by the increased abundance of virulent phages that kill the Lachnospiraceae hosts, and a decreased abundance of temperate phages that piggyback the Bacteroidaceae hosts. Functional profiling reveals an enrichment through horizontal gene transfers of toxin-antitoxin genes on temperate phage genomes in high-leaf season, potentially conferring benefits to their prokaryotic hosts. The phage-host ecological dynamics are driven by the coevolutionary processes which select for tail fiber and DNA primase genes on virulent and temperate phage genomes, respectively. Our results highlight complex phageome-microbiome interactions as a key feature of the gibbon gut microbial ecosystem responding to the seasonal diet.

Identification of a novel adenovirus in liver tissue sample of the Great Himalayan leaf-nosed bat (Hipposideros armiger).

Bats are important reservoirs for many zoonotic viruses. To explore and monitor potential novel viruses carried by bats, 21 liver samples of bats (Hipposideros armiger) were collected from Yunnan Province in southern China. Only one (4.8%) of all models was detected with adenovirus. The whole genome strain obtained by the viral metagenomics method combined with PCR was temporarily named YN01. The complete genome of YN01 was 37,676bp, with a G + C content of 55.20% and 28 open reading frames. Phylogenetic analysis indicated that the strain YN01 can be classified as genus Mastadenovirus and was the most similar to the adenovirus isolated from Rhinolophus sinicus in China in 2016. The analysis is needed to verify the possibility of cross-species transmission. This virological investigation has increased our understanding of the ecology of bat-borne viruses in this area and provided a reference for possible future infectious diseases.

Total metagenomes outperform viromes in recovering viral diversity from sulfuric soils.

Recent metagenomic advancements have offered unprecedented insights into soil viral ecology. However, it remains a challenge to select the suitable metagenomic method for investigating soil viruses under different environmental conditions. Here, we assessed the performance of viral size-fraction metagenomes (viromes) and total metagenomes in capturing viral diversity from hypersulfidic soils with neutral pH and sulfuric soils with pH <3.3. Viromes effectively enhanced the sequencing coverage of viral genomes in both soil types. Viomes of hypersulfidic soils outperformed total metagenomes by recovering a significantly higher number of viral operational taxonomic units (vOTUs). However, total metagenomes of sulfuric soils recovered ~4.5 times more vOTUs than viromes on average. Altogether, our findings suggest that the choice between viromes and total metagenomes for studying soil viruses should be carefully considered based on the specific environmental conditions.

Large-scale single-virus genomics uncovers hidden diversity of river water viruses and diversified gene profiles.

Environmental viruses (primarily bacteriophages) are widely recognized as playing an important role in ecosystem homeostasis through the infection of host cells. However, the majority of environmental viruses are still unknown as their mosaic structure and frequent mutations in their sequences hinder genome construction in current metagenomics. To enable the large-scale acquisition of environmental viral genomes, we developed a new single-viral genome sequencing platform with microfluidic-generated gel beads. Amplification of individual DNA viral genomes in mass-produced gel beads allows high-throughput genome sequencing compared to conventional single-virus genomics. The sequencing analysis of river water samples yielded 1431 diverse viral single-amplified genomes, whereas viral metagenomics recovered 100 viral metagenome-assembled genomes at the comparable sequence depth. The 99.5% of viral single-amplified genomes were determined novel at the species level, most of which could not be recovered by a metagenomic assembly. The large-scale acquisition of diverse viral genomes identified protein clusters commonly detected in different viral strains, allowing the gene transfer to be tracked. Moreover, comparative genomics within the same viral species revealed that the profiles of various methyltransferase subtypes were diverse, suggesting an enhanced escape from host bacterial internal defense mechanisms. Our use of gel bead-based single-virus genomics will contribute to exploring the nature of viruses by accelerating the accumulation of draft genomes of environmental DNA viruses.

CAPTVRED: an automated pipeline for viral tracking and discovery from capture-based metagenomics samples

Abstract Summary Target Enrichment Sequencing or Capture-based metagenomics has emerged as an approach of interest for viral metagenomics in complex samples. However, these datasets are usually analyzed with standard downstream Bioinformatics analyses. CAPTVRED (Capture-based metagenomics Analysis Pipeline for tracking ViRal species from Environmental Datasets), has been designed to assess the virome present in complex samples, specially focused on those obtained by Target Enrichment Sequencing approach. This work aims to provide a user-friendly tool that complements this sequencing approach for the total or partial virome description, especially from environmental matrices. It includes a setup module which allows preparation and adjustment of the pipeline to any capture panel directed to a set of species of interest. The tool also aims to reduce time and computational cost, as well as to provide comprehensive, reproducible, and accessible results while being easy to costume, set up, and install. Availability and implementation Source code and test datasets are freely available at github repository: https://github.com/CompGenLabUB/CAPTVRED.git