Integrating taxonomic signals from MAGs and contigs improves read annotation and taxonomic profiling of metagenomes.

Metagenomic analysis typically includes read-based taxonomic profiling, assembly, and binning of metagenome-assembled genomes (MAGs). Here we integrate these steps in Read Annotation Tool (RAT), which uses robust taxonomic signals from MAGs and contigs to enhance read annotation. RAT reconstructs taxonomic profiles with high precision and sensitivity, outperforming other state-of-the-art tools. In high-diversity groundwater samples, RAT annotates a large fraction of the metagenomic reads, calling novel taxa at the appropriate, sometimes high taxonomic ranks. Thus, RAT integrative profiling provides an accurate and comprehensive view of the microbiome from shotgun metagenomics data. The package of Contig Annotation Tool (CAT), Bin Annotation Tool (BAT), and RAT is available at https://github.com/MGXlab/CAT_pack (from CAT pack v6.0). The CAT pack now also supports Genome Taxonomy Database (GTDB) annotations.

Benchmarking bioinformatic virus identification tools using real-world metagenomic data across biomes.

BACKGROUND: As most viruses remain uncultivated, metagenomics is currently the main method for virus discovery. Detecting viruses in metagenomic data is not trivial. In the past few years, many bioinformatic virus identification tools have been developed for this task, making it challenging to choose the right tools, parameters, and cutoffs. As all these tools measure different biological signals, and use different algorithms and training and reference databases, it is imperative to conduct an independent benchmarking to give users objective guidance. RESULTS: We compare the performance of nine state-of-the-art virus identification tools in thirteen modes on eight paired viral and microbial datasets from three distinct biomes, including a new complex dataset from Antarctic coastal waters. The tools have highly variable true positive rates (0-97%) and false positive rates (0-30%). PPR-Meta best distinguishes viral from microbial contigs, followed by DeepVirFinder, VirSorter2, and VIBRANT. Different tools identify different subsets of the benchmarking data and all tools, except for Sourmash, find unique viral contigs. Performance of tools improved with adjusted parameter cutoffs, indicating that adjustment of parameter cutoffs before usage should be considered. CONCLUSIONS: Together, our independent benchmarking facilitates selecting choices of bioinformatic virus identification tools and gives suggestions for parameter adjustments to viromics researchers.

Taxonomic resolution of different 16S rRNA variable regions varies strongly across plant-associated bacteria.

Plant-microbiome research plays a pivotal role in understanding the relationships between plants and their associated microbial communities, with implications for agriculture and ecosystem dynamics. Metabarcoding analysis on variable regions of the 16S ribosomal RNA (rRNA) gene remains the dominant technology to study microbiome diversity in this field. However, the choice of the targeted variable region might affect the outcome of the microbiome studies. In our in silico analysis, we have evaluated whether the targeted variable region has an impact on taxonomic resolution in 16 plant-related microbial genera. Through a comparison of 16S rRNA gene variable regions with whole-genome data, our findings suggest that the V1-V3 region is generally a more suitable option than the widely used V3-V4 region for targeting microbiome analysis in plant-related genera. However, sole reliance on one region could introduce detection biases for specific genera. Thus, we are suggesting that while transitioning to full-length 16S rRNA gene and whole-genome sequencing for plant-microbiome analysis, the usage of genus-specific variable regions can achieve more precise taxonomic assignments. More broadly, our approach provides a blueprint to identify the most discriminating variable regions of the 16S rRNA gene for genera of interest.

A global survey of host, aquatic, and soil microbiomes reveals shared abundance and genomic features between bacterial and fungal generalists.

Environmental change, coupled with alteration in human lifestyles, is profoundly impacting the microbial communities critical to the health of the Earth and its inhabitants. To identify bacteria and fungi that are resistant and susceptible to habitat change, we analyze thousands of genera detected in 1,580 host, soil, and aquatic samples. This large-scale analysis identifies 48 bacterial and 4 fungal genera that are abundant across the three biomes, demonstrating fitness in diverse environmental conditions. Samples containing these generalists have significantly higher alpha diversity. These generalists play a significant role in shaping cross-kingdom community structure, boasting larger genomes with more secondary metabolism and antimicrobial resistance genes. Conversely, 30 bacterial and 19 fungal genera are only found in a single habitat, suggesting a limited ability to adapt to different and changing environments. These findings contribute to our understanding of microbial niche breadth and its consequences for global biodiversity loss.

Ultraconserved bacteriophage genome sequence identified in 1300-year-old human palaeofaeces.

Bacteriophages are widely recognised as rapidly evolving biological entities. However, knowledge about ancient bacteriophages is limited. Here, we analyse DNA sequence datasets previously generated from ancient palaeofaeces and human gut-content samples, and identify an ancient phage genome nearly identical to present-day Mushuvirus mushu, a virus that infects gut commensal bacteria. The DNA damage patterns of the genome are consistent with its ancient origin and, despite 1300 years of evolution, the ancient Mushuvirus genome shares 97.7% nucleotide identity with its modern counterpart, indicating a long-term relationship between the prophage and its host. In addition, we reconstruct and authenticate 297 other phage genomes from the last 5300 years, including those belonging to unknown families. Our findings demonstrate the feasibility of reconstructing ancient phage genome sequences, thus expanding the known virosphere and offering insights into phage-bacteria interactions spanning several millennia.

Identifying and tracking mobile elements in evolving compost communities yields insights into the nanobiome.

Microbial evolution is driven by rapid changes in gene content mediated by horizontal gene transfer (HGT). While mobile genetic elements (MGEs) are important drivers of gene flux, the nanobiome-the zoo of Darwinian replicators that depend on microbial hosts-remains poorly characterised. New approaches are necessary to increase our understanding beyond MGEs shaping individual populations, towards their impacts on complex microbial communities. A bioinformatic pipeline (xenoseq) was developed to cross-compare metagenomic samples from microbial consortia evolving in parallel, aimed at identifying MGE dissemination, which was applied to compost communities which underwent periodic mixing of MGEs. We show that xenoseq can distinguish movement of MGEs from demographic changes in community composition that otherwise confounds identification, and furthermore demonstrate the discovery of various unexpected entities. Of particular interest was a nanobacterium of the candidate phylum radiation (CPR) which is closely related to a species identified in groundwater ecosystems (Candidatus Saccharibacterium), and appears to have a parasitic lifestyle. We also highlight another prolific mobile element, a 313 kb plasmid hosted by a Cellvibrio lineage. The host was predicted to be capable of nitrogen fixation, and acquisition of the plasmid coincides with increased ammonia production. Taken together, our data show that new experimental strategies combined with bioinformatic analyses of metagenomic data stand to provide insight into the nanobiome as a driver of microbial community evolution.

Changes to virus taxonomy and the ICTV Statutes ratified by the International Committee on Taxonomy of Viruses (2023).

This article reports changes to virus taxonomy and taxon nomenclature that were approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in April 2023. The entire ICTV membership was invited to vote on 174 taxonomic proposals that had been approved by the ICTV Executive Committee in July 2022, as well as a proposed revision of the ICTV Statutes. All proposals and the revised ICTV Statutes were approved by a majority of the voting membership. Of note, the ICTV continued the process of renaming existing species in accordance with the recently mandated binomial format and included gene transfer agents (GTAs) in the classification framework by classifying them as viriforms. In total, one class, seven orders, 31 families, 214 genera, and 858 species were created.

Growth rate is a dominant factor predicting the rhizosphere effect.

The root microbiome is shaped by plant root activity, which selects specific microbial taxa from the surrounding soil. This influence on the microorganisms and soil chemistry in the immediate vicinity of the roots has been referred to as the rhizosphere effect. Understanding the traits that make bacteria successful in the rhizosphere is critical for developing sustainable agriculture solutions. In this study, we compared the growth rate potential, a complex trait that can be predicted from bacterial genome sequences, to functional traits encoded by proteins. We analyzed 84 paired rhizosphere- and soil-derived 16S rRNA gene amplicon datasets from 18 different plants and soil types, performed differential abundance analysis, and estimated growth rates for each bacterial genus. We found that bacteria with higher growth rate potential consistently dominated the rhizosphere, and this trend was confirmed in different bacterial phyla using genome sequences of 3270 bacterial isolates and 6707 metagenome-assembled genomes (MAGs) from 1121 plant- and soil-associated metagenomes. We then identified which functional traits were enriched in MAGs according to their niche or growth rate status. We found that predicted growth rate potential was the main feature for differentiating rhizosphere and soil bacteria in machine learning models, and we then analyzed the features that were important for achieving faster growth rates, which makes bacteria more competitive in the rhizosphere. As growth rate potential can be predicted from genomic data, this work has implications for understanding bacterial community assembly in the rhizosphere, where many uncultivated bacteria reside.

Bacterial Oncotraits Rather than Spatial Organization Are Associated with Dysplasia in Ulcerative Colitis.

BACKGROUND AND AIMS: Colonic bacterial biofilms are frequently present in ulcerative colitis [UC] and may increase dysplasia risk through pathogens expressing oncotraits. This prospective cohort study aimed to determine [1] the association of oncotraits and longitudinal biofilm presence with dysplasia risk in UC, and [2] the relation of bacterial composition with biofilms and dysplasia risk. METHODS: Faeces and left- and right-sided colonic biopsies were collected from 80 UC patients and 35 controls. Oncotraits [FadA of Fusobacterium, BFT of Bacteroides fragilis, colibactin [ClbB] and Intimin [Eae] of Escherichia coli] were assessed in faecal DNA with multiplex quantitative polymerase chain reaction [qPCR]. Biopsies were screened for biofilms [n = 873] with 16S rRNA fluorescent in situ hybridiation. Shotgun metagenomic sequencing [n = 265], and ki67-immunohistochemistry were performed. Associations were determined with a mixed-effects regression model. RESULTS: Biofilms were highly prevalent in UC patients [90.8%] with a median persistence of 3 years (interquartile range [IQR] 2-5 years). Biofilm-positive biopsies showed increased epithelial hypertrophy [p = 0.025] and a reduced Shannon diversity independent of disease status [p = 0.015], but were not significantly associated with dysplasia in UC: adjusted odds ratio [aOR] 1.45, 95% confidence interval [CI] 0.63-3.40. In contrast, ClbB independently associated with dysplasia [aOR 7.16, 95% CI 1.75-29.28], and FadA and Fusobacteriales were associated with a decreased dysplasia risk in UC [aOR 0.23, 95% CI 0.06-0.83, p <0.01]. CONCLUSIONS: Biofilms are a hallmark of UC; however, because of their high prevalence are a poor biomarker for dysplasia. In contrast, colibactin presence and FadA absence independently associate with dysplasia in UC and might therefore be valuable biomarkers for future risk stratification and intervention strategies.

Virus taxonomy and the role of the International Committee on Taxonomy of Viruses (ICTV).

The taxonomy of viruses is developed and overseen by the International Committee on Taxonomy of Viruses (ICTV), which scrutinizes, approves and ratifies taxonomic proposals, and maintains a list of virus taxa with approved names (https://ictv.global). The ICTV has approximately 180 members who vote by simple majority. Taxon-specific Study Groups established by the ICTV have a combined membership of over 600 scientists from the wider virology community; they provide comprehensive expertise across the range of known viruses and are major contributors to the creation and evaluation of taxonomic proposals. Proposals can be submitted by anyone and will be considered by the ICTV irrespective of Study Group support. Thus, virus taxonomy is developed from within the virology community and realized by a democratic decision-making process. The ICTV upholds the distinction between a virus or replicating genetic element as a physical entity and the taxon category to which it is assigned. This is reflected by the nomenclature of the virus species taxon, which is now mandated by the ICTV to be in a binomial format (genus + species epithet) and is typographically distinct from the names of viruses. Classification of viruses below the rank of species (such as, genotypes or strains) is not within the remit of the ICTV. This article, authored by the ICTV Executive Committee, explains the principles of virus taxonomy and the organization, function, processes and resources of the ICTV, with the aim of encouraging greater understanding and interaction among the wider virology community.

iPHoP: An integrated machine learning framework to maximize host prediction for metagenome-derived viruses of archaea and bacteria.

The extraordinary diversity of viruses infecting bacteria and archaea is now primarily studied through metagenomics. While metagenomes enable high-throughput exploration of the viral sequence space, metagenome-derived sequences lack key information compared to isolated viruses, in particular host association. Different computational approaches are available to predict the host(s) of uncultivated viruses based on their genome sequences, but thus far individual approaches are limited either in precision or in recall, i.e., for a number of viruses they yield erroneous predictions or no prediction at all. Here, we describe iPHoP, a two-step framework that integrates multiple methods to reliably predict host taxonomy at the genus rank for a broad range of viruses infecting bacteria and archaea, while retaining a low false discovery rate. Based on a large dataset of metagenome-derived virus genomes from the IMG/VR database, we illustrate how iPHoP can provide extensive host prediction and guide further characterization of uncultivated viruses.

A social niche breadth score reveals niche range strategies of generalists and specialists.

Generalists can survive in many environments, whereas specialists are restricted to a single environment. Although a classical concept in ecology, niche breadth has remained challenging to quantify for microorganisms because it depends on an objective definition of the environment. Here, by defining the environment of a microorganism as the community it resides in, we integrated information from over 22,000 environmental sequencing samples to derive a quantitative measure of the niche, which we call social niche breadth. At the level of genera, we explored niche range strategies throughout the prokaryotic tree of life. We found that social generalists include opportunists that stochastically dominate local communities, whereas social specialists are stable but low in abundance. Social generalists have a more diverse and open pan-genome than social specialists, but we found no global correlation between social niche breadth and genome size. Instead, we observed two distinct evolutionary strategies, whereby specialists have relatively small genomes in habitats with low local diversity, but relatively large genomes in habitats with high local diversity. Together, our analysis shines data-driven light on microbial niche range strategies.

Four principles to establish a universal virus taxonomy.

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., "arboviruses"), infecting a certain type of host (e.g., "mycoviruses," "bacteriophages") or displaying specific pathogenicity (e.g., "human immunodeficiency viruses"), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent.

Rational Design of Profile HMMs for Sensitive and Specific Sequence Detection with Case Studies Applied to Viruses, Bacteriophages, and Casposons.

Profile hidden Markov models (HMMs) are a powerful way of modeling biological sequence diversity and constitute a very sensitive approach to detecting divergent sequences. Here, we report the development of protocols for the rational design of profile HMMs. These methods were implemented on TABAJARA, a program that can be used to either detect all biological sequences of a group or discriminate specific groups of sequences. By calculating position-specific information scores along a multiple sequence alignment, TABAJARA automatically identifies the most informative sequence motifs and uses them to construct profile HMMs. As a proof-of-principle, we applied TABAJARA to generate profile HMMs for the detection and classification of two viral groups presenting different evolutionary rates: bacteriophages of the Microviridae family and viruses of the Flavivirus genus. We obtained conserved models for the generic detection of any Microviridae or Flavivirus sequence, and profile HMMs that can specifically discriminate Microviridae subfamilies or Flavivirus species. In another application, we constructed Cas1 endonuclease-derived profile HMMs that can discriminate CRISPRs and casposons, two evolutionarily related transposable elements. We believe that the protocols described here, and implemented on TABAJARA, constitute a generic toolbox for generating profile HMMs for the highly sensitive and specific detection of sequence classes.

Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Recent changes to virus taxonomy ratified by the International Committee on Taxonomy of Viruses (2022).

This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2022. The entire ICTV was invited to vote on 174 taxonomic proposals approved by the ICTV Executive Committee at its annual meeting in July 2021. All proposals were ratified by an absolute majority of the ICTV members. Of note, the Study Groups have started to implement the new rule for uniform virus species naming that became effective in 2021 and mandates the binomial 'Genus_name species_epithet' format with or without Latinization. As a result of this ratification, the names of 6,481 virus species (more than 60 percent of all species names currently recognized by ICTV) now follow this format.

Nutrition or nature: using elementary flux modes to disentangle the complex forces shaping prokaryote pan-genomes.

BACKGROUND: Microbial pan-genomes are shaped by a complex combination of stochastic and deterministic forces. Even closely related genomes exhibit extensive variation in their gene content. Understanding what drives this variation requires exploring the interactions of gene products with each other and with the organism's external environment. However, to date, conceptual models of pan-genome dynamics often represent genes as independent units and provide limited information about their mechanistic interactions. RESULTS: We simulated the stochastic process of gene-loss using the pooled genome-scale metabolic reaction networks of 46 taxonomically diverse bacterial and archaeal families as proxies for their pan-genomes. The frequency by which reactions are retained in functional networks when stochastic gene loss is simulated in diverse environments allowed us to disentangle the metabolic reactions whose presence depends on the metabolite composition of the external environment (constrained by "nutrition") from those that are independent of the environment (constrained by "nature"). By comparing the frequency of reactions from the first group with their observed frequencies in bacterial and archaeal families, we predicted the metabolic niches that shaped the genomic composition of these lineages. Moreover, we found that the lineages that were shaped by a more diverse metabolic niche also occur in more diverse biomes as assessed by global environmental sequencing datasets. CONCLUSION: We introduce a computational framework for analyzing and interpreting pan-reactomes that provides novel insights into the ecological and evolutionary drivers of pan-genome dynamics.