Evaluation of viral genome assembly and diversity estimation in deep metagenomes.

BACKGROUND: Viruses have unique properties, small genome and regions of high similarity, whose effects on metagenomic assemblies have not been characterized so far. This study uses diverse in silico simulated viromes to evaluate how extensively genomes can be assembled using different sequencing platforms and assemblers. Further, it investigates the suitability of different methods to estimate viral diversity in metagenomes. RESULTS: We created in silico metagenomes mimicking various platforms at different sequencing depths. The CLC assembler revealed subpar compared to IDBA_UD and CAMERA , which are metagenomic-specific. Up to a saturation point, Illumina platforms proved more capable of reconstructing large portions of viral genomes compared to 454. Read length was an important factor for limiting chimericity, while scaffolding marginally improved contig length and accuracy. The genome length of the various viruses in the metagenomes did not significantly affect genome reconstruction, but the co-existence of highly similar genomes was detrimental. When evaluating diversity estimation tools, we found that PHACCS results were more accurate than those from CatchAll and clustering, which were both orders of magnitude above expected. CONCLUSIONS: Assemblers designed specifically for the analysis of metagenomes should be used to facilitate the creation of high-quality long contigs. Despite the high coverage possible, scientists should not expect to always obtain complete genomes, because their reconstruction may be hindered by co-existing species bearing highly similar genomic regions. Further development of metagenomics-oriented assemblers may help bypass these limitations in future studies. Meanwhile, the lack of fully reconstructed communities keeps methods to estimate viral diversity relevant. While none of the three methods tested had absolute precision, only PHACCS was deemed suitable for comparative studies.

Genome sequences of two Arthrobacter phages isolated from soil.

The isolation and characterization of additional phages is crucial for adding reliable viral sequences with relevant biological information to viral databases. In this study, we present the complete genomes of two Arthrobacter phages obtained from different soil samples.

Draft genome sequence of Arthrobacter sp. strain B10-11 isolated from the tomato rhizosphere.

In the present work, we present the draft genome sequence of a new putative Arthrobacter species associated with the tomato rhizosphere.

Zoonotic potential of urban wildlife faeces, assessed through metabarcoding.

Monitoring zoonoses in urban environments is of great relevance, where the incidence of certain pathogens may be higher and where population density makes the spread of any contagious disease more likely. In this study we applied a metabarcoding approach to study potentially zoonotic pathogens in faecal samples of 9 urban vertebrate species. We applied this methodology with two objectives. Firstly, to obtain information on potential pathogens present in the urban fauna of a large European city (Madrid, Spain) and to determine which are their main reservoirs. In addition, we tested for differences in the prevalence of these potential pathogens between urban and rural European rabbits, used as ubiquitous species. Additionally, based on the results obtained, we evaluated the effectiveness of metabarcoding as a tool for monitoring potential pathogen. Our results revealed the presence of potentially zoonotic bacterial genera in all studied host species, 10 of these genera with zoonotic species of mandatory monitoring in the European Union. Based on these results, urban birds (especially house sparrows and pigeons) and bats are the species posing the greatest potential risk, with Campylobacter and Listeria genera in birds and of Chlamydia and Vibrio cholerae in bats as most relevant pathogens. This information highlights the risk associated with fresh faeces from urban wildlife. In addition, we detected Campylobacter in >50 % of the urban rabbit samples, while we only detected it in 11 % of the rural rabbit samples. We found that urban rabbits have a higher prevalence of some pathogens relative to rural rabbits, which could indicate increased risk of pathogen transmission to humans. Finally, our results showed that metabarcoding can be an useful tool to quickly obtain a first screening of potentially zoonotic organisms, necessary information to target the monitoring efforts on the most relevant pathogens and host species.

Leveraging phylogenetic signal to unravel microbiome function and assembly rules.

Clarifying the general rules behind microbial community assembly will foster the development of microbiome-based technological solutions. Here, we study microbial community assembly through a computational analysis of phylogenetic core groups (PCGs): discrete portions of the bacterial phylogeny with high prevalence in the ecosystem under study. We first show that the existence of PCGs was a predominant feature of the varied set of microbial ecosystems studied. Then, we re-analyzed an in vitro experimental dataset using a PCG-based approach, drawing only from its community composition data and from publicly available genomic databases. Using mainly genome scale metabolic models and population dynamics modeling, we obtained ecological insights on metabolic niche structure and population dynamics comparable to those gained after canonical experimentation. Thus, leveraging phylogenetic signal to help unravel microbiome function and assembly rules offers a potential avenue to gain further insight on Earth's microbial ecosystems.

The effects from DNA extraction methods on the evaluation of microbial diversity associated with human colonic tissue.

Potentially valuable sources of DNA have been extracted from human colonic tissues and are retained in biobanks throughout the world, and might be re-examined to better understand host-microbe interactions in health and disease. However, the published protocols for DNA extraction typically used by gastroenterologists have not been systematically compared in terms of their recovery of the microbial fraction associated with colonic tissue. For this reason, we examined how three different tissue DNA extraction methods (the QIAGEN AllPrep DNA/RNA kit, salting out and high molecular weight (HMW) methods of DNA extraction) employed in past clinical trials, and the repeated bead beating and column (RBB+C) method might impact the recovery of microbial DNA from colonic tissue, using a custom designed phylogenetic microarray for gut bacteria and archaea. All four methods produced very similar profiles of the microbial diversity, but there were some differences in probe signal intensities, with the HMW method producing stronger probe intensities for a subset of the Firmicutes probes including Clostridium and Streptococcus spp. Real-time PCR analysis revealed that the HMW and RBB+C extracted DNA contained significantly more DNA of Firmicutes origin and that the different DNA extraction methods also gave variable results in terms of host DNA recovery. All of the methods tested recovered DNA from the archaeal community although there were some differences in probe signal intensity. Based on these findings, we conclude that while all four methods are efficacious at releasing microbial DNA from biopsy tissue samples, the HMW and RBB+C methods of DNA extraction may release more DNA from some of the Firmicutes bacteria associated with colonic tissue. Thus, DNA archived in biobanks could be suitable for retrospective profiling analyses, provided the caveats with respect to the DNA extraction method(s) used are taken into account.

Assessment of phylo-functional coherence along the bacterial phylogeny and taxonomy.

In this report we use available curated phylogenies, taxonomy, and genome annotations to assess the phylogenetic and gene content similarity associated with each different taxon and taxonomic rank. Subsequently, we employ the same data to assess the frontiers of functional coherence along the bacterial phylogeny. Our results show that within-group phylogenetic and gene content similarity of taxa in the same rank are not homogenous, and that these values show extensive overlap between ranks. Functional coherence along the 16S rRNA gene-based phylogeny was limited to 44 particular nodes presenting large variations in phylogenetic depth. For instance, the deep subtree affiliated to class Actinobacteria presented functional coherence, while the shallower family Enterobacteriaceae-affiliated subtree did not. On the other hand, functional coherence along the genome-based phylogeny delimited deep subtrees affiliated to phyla Actinobacteriota, Deinococcota, Chloroflexota, Firmicutes, and a subtree containing the rest of the bacterial phyla. The results presented here can be used to guide the exploration of results in many microbial ecology and evolution research scenarios. Moreover, we provide dedicated scripts and files that can be used to continue the exploration of functional coherence along the bacterial phylogeny employing different parameters or input data ( https://git.io/Jec5U ).

Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable to form biofilms on abiotic surfaces.

Motility is a key trait for rhizosphere colonization by Pseudomonas fluorescens. Mutants with reduced motility are poor competitors, and hypermotile, more competitive phenotypic variants are selected in the rhizosphere. Flagellar motility is a feature associated to planktonic, free-living single cells, and although it is necessary for the initial steps of biofilm formation, bacteria in biofilm lack flagella. To test the correlation between biofilm formation and rhizosphere colonization, we have used P. fluorescens F113 hypermotile derivatives and mutants affected in regulatory genes which in other bacteria modulate biofilm development, namely gacS (G), sadB (S) and wspR (W). Mutants affected in these three genes and a hypermotile variant (V35) isolated from the rhizosphere were impaired in biofilm formation on abiotic surfaces, but colonized the alfalfa root apex as efficiently as the wild-type strain, indicating that biofilm formation on abiotic surfaces and rhizosphere colonization follow different regulatory pathways in P. fluorescens. Furthermore, a triple mutant gacSsadBwspR (GSW) and V35 were more competitive than the wild-type strain for root-tip colonization, suggesting that motility is more relevant in this environment than the ability to form biofilms on abiotic surfaces. Microscopy showed the same root colonization pattern for P. fluorescens F113 and all the derivatives: extensive microcolonies, apparently held to the rhizoplane by a mucigel that seems to be plant produced. Therefore, the ability to form biofilms on abiotic surfaces does not necessarily correlates with efficient rhizosphere colonization or competitive colonization.

Experimental and computational approaches to unravel microbial community assembly.

Microbial communities have a preponderant role in the life support processes of our common home planet Earth. These extremely diverse communities drive global biogeochemical cycles, and develop intimate relationships with most multicellular organisms, with a significant impact on their fitness. Our understanding of their composition and function has enjoyed a significant thrust during the last decade thanks to the rise of high-throughput sequencing technologies. Intriguingly, the diversity patterns observed in nature point to the possible existence of fundamental community assembly rules. Unfortunately, these rules are still poorly understood, despite the fact that their knowledge could spur a scientific, technological, and economic revolution, impacting, for instance, agricultural, environmental, and health-related practices. In this minireview, I recapitulate the most important wet lab techniques and computational approaches currently employed in the study of microbial community assembly, and briefly discuss various experimental designs. Most of these approaches and considerations are also relevant to the study of microbial microevolution, as it has been shown that it can occur in ecological relevant timescales. Moreover, I provide a succinct review of various recent studies, chosen based on the diversity of ecological concepts addressed, experimental designs, and choice of wet lab and computational techniques. This piece aims to serve as a primer to those new to the field, as well as a source of new ideas to the more experienced researchers.

A comprehensive human minimal gut metagenome extends the host's metabolic potential.

Accumulating evidence suggests that humans could be considered as holobionts in which the gut microbiota play essential functions. Initial metagenomic studies reported a pattern of shared genes in the gut microbiome of different individuals, leading to the definition of the minimal gut metagenome as the set of microbial genes necessary for homeostasis and present in all healthy individuals. This study analyses the minimal gut metagenome of the most comprehensive dataset available, including individuals from agriculturalist and industrialist societies, also embodying highly diverse ethnic and geographical backgrounds. The outcome, based on metagenomic predictions for community composition data, resulted in a minimal metagenome comprising 3412 genes, mapping to 1856 reactions and 128 metabolic pathways predicted to occur across all individuals. These results were substantiated by the analysis of two additional datasets describing the microbial community compositions of larger Western cohorts, as well as a substantial shotgun metagenomics dataset. Subsequent analyses showed the plausible metabolic complementarity provided by the minimal gut metagenome to the human genome.