The human body hosts vast microbial communities, termed the microbiome. Less well known is the fact that the human body also hosts vast numbers of different viruses, collectively termed the 'virome'. ...Viruses are believed to be the most abundant and diverse biological entities on our planet, with an estimated 10
particles on Earth. The human virome is similarly vast and complex, consisting of approximately 10
particles per human individual, with great heterogeneity. In recent years, studies of the human virome using metagenomic sequencing and other methods have clarified aspects of human virome diversity at different body sites, the relationships to disease states and mechanisms of establishment of the human virome during early life. Despite increasing focus, it remains the case that the majority of sequence data in a typical virome study remain unidentified, highlighting the extent of unexplored viral 'dark matter'. Nevertheless, it is now clear that viral community states can be associated with adverse outcomes for the human host, whereas other states are characteristic of health. In this Review, we provide an overview of research on the human virome and highlight outstanding recent studies that explore the assembly, composition and dynamics of the human virome as well as host-virome interactions in health and disease.
Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential for ...a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples. We generated influenza virus reads down to a limit of detection of 10
to 10
genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (
= 4.7 × 10
). Applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold
values, <30) and 6/13 samples with low viral titers (
values, 30 to 40). No false-positive reads were generated from 10 influenza virus-negative samples. Thus, Nanopore sequencing operated with 83% sensitivity (95% confidence interval CI, 67 to 93%) and 100% specificity (95% CI, 69 to 100%) compared to the current diagnostic standard. Coverage of full-length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza virus titers, we were able to reconstruct >99% complete sequences for all eight gene segments. We also detected a human coronavirus coinfection in one clinical sample. While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses.
The acquisition and development of the infant microbiome are key to establishing a healthy host-microbiome symbiosis. The maternal microbial reservoir is thought to play a crucial role in this ...process. However, the source and transmission routes of the infant pioneering microbes are poorly understood. To address this, we longitudinally sampled the microbiome of 25 mother-infant pairs across multiple body sites from birth up to 4 months postpartum. Strain-level metagenomic profiling showed a rapid influx of microbes at birth followed by strong selection during the first few days of life. Maternal skin and vaginal strains colonize only transiently, and the infant continues to acquire microbes from distinct maternal sources after birth. Maternal gut strains proved more persistent in the infant gut and ecologically better adapted than those acquired from other sources. Together, these data describe the mother-to-infant microbiome transmission routes that are integral in the development of the infant microbiome.
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•Strain-resolved metagenomics was used to track mother-to-infant microbiome transfer•Microbial strains from multiple maternal body sites transfer to the infant microbiome•The early microbial diversity in the infant gut is rapidly shaped by niche selection•The maternal gut microbiome is the source of the majority of transmitted strains
Ferretti et al. use metagenomics with strain-resolved computational profiling to characterize the transfer of microbes from mothers to their infants during their first 4 months of life. Multiple maternal body sites contribute to the developing infant microbiome, with maternal gut strains providing the largest contribution of colonizing microorganisms.
Recently, sequencing technologies have become readily available, and scientists are more motivated to conduct metagenomic research to unveil the potential of a myriad of ecosystems and biomes. ...Metagenomics studies the composition and functions of microbial communities and paves the way to multiple applications in medicine, industry, and ecology. Nonetheless, the immense amount of sequencing data of metagenomics research and the few user-friendly analysis tools and pipelines carry a new challenge to the data analysis.Web-based bioinformatics tools are now being developed to facilitate the analysis of complex metagenomic data without prior knowledge of any programming languages or special installation. Specialized web tools help answer researchers' main questions on the taxonomic classification, functional capabilities, discrepancies between two ecosystems, and the probable functional correlations between the members of a specific microbial community. With an Internet connection and a few clicks, researchers can conveniently and efficiently analyze the metagenomic datasets, summarize results, and visualize key information on the composition and the functional potential of metagenomic samples under study. This chapter provides a simple guide to a few of the fundamental web-based services used for metagenomic data analyses, such as BV-BRC, RDP, MG-RAST, MicrobiomeAnalyst, METAGENassist, and MGnify.
Current estimates suggest that even in the most resourced settings, the aetiology of encephalitis is identified in less than half of clinical cases. It is acknowledged that filling this gap needs a ...combination of rigorous sampling and improved diagnostic technologies. Next generation sequencing (NGS) methods are powerful tools with the potential for comprehensive and unbiased detection of pathogens in clinical samples. We reviewed the use of this new technology for the diagnosis of suspected infectious encephalitis, and discuss the feasibility for introduction of NGS methods as a frontline diagnostic test.
A systematic literature review was performed, using MESH and text word searches for variants of “sequencing” and “encephalitis” in Medline and EMbase, and searching bibliographies and citations using the Web of Science database. Two authors independently reviewed, extracted and summarised data.
The review identified 25 articles reporting 44 case reports of patients with suspected encephalitis for whom NGS was used as a diagnostic tool. We present the data and highlight themes arising from these cases. There are no randomly controlled trials to assess the utility of NGS as a diagnostic tool.
There is increasing evidence of a role for NGS in the work-up of undiagnosed encephalitis. Lower costs and increasing accessibility of these technologies will facilitate larger studies of these patients. We recommend NGS should be considered as a front-line diagnostic test in chronic and recurring presentations and, given current sample-to-result turn-around times, as second-line in acute cases of encephalitis.
Long sequencing reads are information-rich: aiding de novo assembly and reference mapping, and consequently have great potential for the study of microbial communities. However, the best approaches ...for analysis of long-read metagenomic data are unknown. Additionally, rigorous evaluation of bioinformatics tools is hindered by a lack of long-read data from validated samples with known composition.
We sequenced 2 commercially available mock communities containing 10 microbial species (ZymoBIOMICS Microbial Community Standards) with Oxford Nanopore GridION and PromethION. Both communities and the 10 individual species isolates were also sequenced with Illumina technology. We generated 14 and 16 gigabase pairs from 2 GridION flowcells and 150 and 153 gigabase pairs from 2 PromethION flowcells for the evenly distributed and log-distributed communities, respectively. Read length N50 ranged between 5.3 and 5.4 kilobase pairs over the 4 sequencing runs. Basecalls and corresponding signal data are made available (4.2 TB in total). Alignment to Illumina-sequenced isolates demonstrated the expected microbial species at anticipated abundances, with the limit of detection for the lowest abundance species below 50 cells (GridION). De novo assembly of metagenomes recovered long contiguous sequences without the need for pre-processing techniques such as binning.
We present ultra-deep, long-read nanopore datasets from a well-defined mock community. These datasets will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and comparison of current laboratory and software pipelines.
Advances in bioinformatics recently allowed for the recovery of 'metagenomes assembled genomes' from human microbiome studies carried on with shotgun sequencing techniques. Such approach is used as a ...mean to discover new unclassified metagenomic species, putative biological entities having distinct metabolic traits.
In the present analysis we compare 400 genomes from isolates available on NCBI database and 10,000 human gut metagenomic species, screening all of them for the presence of a minimal set of core functionalities necessary, but not sufficient, for life. As a result, the metagenome-assembled genomes resulted systematically depleted in genes encoding for essential functions apparently needed to support autonomous bacterial life.
The relevant degree of lacking core functionalities that we observed in metagenome-assembled genomes raises some concerns about the effective completeness of metagenome-assembled genomes, suggesting caution in extrapolating biological information about their metabolic propensity and ecology in a complex environment like the human gastrointestinal tract.
Mash extends the MinHash dimensionality-reduction technique to include a pairwise mutation distance and P value significance test, enabling the efficient clustering and search of massive sequence ...collections. Mash reduces large sequences and sequence sets to small, representative sketches, from which global mutation distances can be rapidly estimated. We demonstrate several use cases, including the clustering of all 54,118 NCBI RefSeq genomes in 33 CPU h; real-time database search using assembled or unassembled Illumina, Pacific Biosciences, and Oxford Nanopore data; and the scalable clustering of hundreds of metagenomic samples by composition. Mash is freely released under a BSD license ( https://github.com/marbl/mash ).
EnteroBase is an integrated software environment that supports the identification of global population structures within several bacterial genera that include pathogens. Here, we provide an overview ...of how EnteroBase works, what it can do, and its future prospects. EnteroBase has currently assembled more than 300,000 genomes from Illumina short reads from
,
,
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,
, and
and genotyped those assemblies by core genome multilocus sequence typing (cgMLST). Hierarchical clustering of cgMLST sequence types allows mapping a new bacterial strain to predefined population structures at multiple levels of resolution within a few hours after uploading its short reads. Case Study 1 illustrates this process for local transmissions of
serovar Agama between neighboring social groups of badgers and humans. EnteroBase also supports single nucleotide polymorphism (SNP) calls from both genomic assemblies and after extraction from metagenomic sequences, as illustrated by Case Study 2 which summarizes the microevolution of
over the last 5000 years of pandemic plague. EnteroBase can also provide a global overview of the genomic diversity within an entire genus, as illustrated by Case Study 3, which presents a novel, global overview of the population structure of all of the species, subspecies, and clades within
.
Advances in sequencing technologies and bioinformatics tools have dramatically increased the recovery rate of microbial genomes from metagenomic data. Assessing the quality of metagenome-assembled ...genomes (MAGs) is a critical step before downstream analysis. Here, we present CheckM2, an improved method of predicting genome quality of MAGs using machine learning. Using synthetic and experimental data, we demonstrate that CheckM2 outperforms existing tools in both accuracy and computational speed. In addition, CheckM2's database can be rapidly updated with new high-quality reference genomes, including taxa represented only by a single genome. We also show that CheckM2 accurately predicts genome quality for MAGs from novel lineages, even for those with reduced genome size (for example, Patescibacteria and the DPANN superphylum). CheckM2 provides accurate genome quality predictions across bacterial and archaeal lineages, giving increased confidence when inferring biological conclusions from MAGs.
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