Massively parallel pyrosequencing of the small subunit (16S) ribosomal RNA gene has revealed that the extent of rare microbial populations in several environments, the 'rare biosphere', is orders of ...magnitude higher than previously thought. One important caveat with this method is that sequencing error could artificially inflate diversity estimates. Although the per-base error of 16S rDNA amplicon pyrosequencing has been shown to be as good as or lower than Sanger sequencing, no direct assessments of pyrosequencing errors on diversity estimates have been reported. Using only Escherichia coli MG1655 as a reference template, we find that 16S rDNA diversity is grossly overestimated unless relatively stringent read quality filtering and low clustering thresholds are applied. In particular, the common practice of removing reads with unresolved bases and anomalous read lengths is insufficient to ensure accurate estimates of microbial diversity. Furthermore, common and reproducible homopolymer length errors can result in relatively abundant spurious phylotypes further confounding data interpretation. We suggest that stringent quality-based trimming of 16S pyrotags and clustering thresholds no greater than 97% identity should be used to avoid overestimates of the rare biosphere.
Abstract
The Genome Taxonomy Database (GTDB; https://gtdb.ecogenomic.org) provides a phylogenetically consistent and rank normalized genome-based taxonomy for prokaryotic genomes sourced from the ...NCBI Assembly database. GTDB R06-RS202 spans 254 090 bacterial and 4316 archaeal genomes, a 270% increase since the introduction of the GTDB in November, 2017. These genomes are organized into 45 555 bacterial and 2339 archaeal species clusters which is a 200% increase since the integration of species clusters into the GTDB in June, 2019. Here, we explore prokaryotic diversity from the perspective of the GTDB and highlight the importance of metagenome-assembled genomes in expanding available genomic representation. We also discuss improvements to the GTDB website which allow tracking of taxonomic changes, easy assessment of genome assembly quality, and identification of genomes assembled from type material or used as species representatives. Methodological updates and policy changes made since the inception of the GTDB are then described along with the procedure used to update species clusters in the GTDB. We conclude with a discussion on the use of average nucleotide identities as a pragmatic approach for delineating prokaryotic species.
STAMP is a graphical software package that provides statistical hypothesis tests and exploratory plots for analysing taxonomic and functional profiles. It supports tests for comparing pairs of ...samples or samples organized into two or more treatment groups. Effect sizes and confidence intervals are provided to allow critical assessment of the biological relevancy of test results. A user-friendly graphical interface permits easy exploration of statistical results and generation of publication-quality plots.
STAMP is licensed under the GNU GPL. Python source code and binaries are available from our website at: http://kiwi.cs.dal.ca/Software/STAMP.
The Genome Taxonomy Database is a phylogenetically consistent, genome-based taxonomy that provides rank-normalized classifications for ~150,000 bacterial and archaeal genomes from domain to genus. ...However, almost 40% of the genomes in the Genome Taxonomy Database lack a species name. We address this limitation by using commonly accepted average nucleotide identity criteria to set bounds on species and propose species clusters that encompass all publicly available bacterial and archaeal genomes. Unlike previous average nucleotide identity studies, we chose a single representative genome to serve as the effective nomenclatural 'type' defining each species. Of the 24,706 proposed species clusters, 8,792 are based on published names. We assigned placeholder names to the remaining 15,914 species clusters to provide names to the growing number of genomes from uncultivated species. This resource provides a complete domain-to-species taxonomic framework for bacterial and archaeal genomes, which will facilitate research on uncultivated species and improve communication of scientific results.
Arrays of clustered, regularly interspaced short palindromic repeats (CRISPRs) are widespread in the genomes of many bacteria and almost all archaea. These arrays are composed of direct repeats that ...are separated by similarly sized non-repetitive spacers. CRISPR arrays, together with a group of associated proteins, confer resistance to phages, possibly by an RNA-interference-like mechanism. This Progress discusses the structure and function of this newly recognized antiviral mechanism.
Comprehensive, high-quality reference genomes are required for functional characterization and taxonomic assignment of the human gut microbiota. We present the Unified Human Gastrointestinal Genome ...(UHGG) collection, comprising 204,938 nonredundant genomes from 4,644 gut prokaryotes. These genomes encode >170 million protein sequences, which we collated in the Unified Human Gastrointestinal Protein (UHGP) catalog. The UHGP more than doubles the number of gut proteins in comparison to those present in the Integrated Gene Catalog. More than 70% of the UHGG species lack cultured representatives, and 40% of the UHGP lack functional annotations. Intraspecies genomic variation analyses revealed a large reservoir of accessory genes and single-nucleotide variants, many of which are specific to individual human populations. The UHGG and UHGP collections will enable studies linking genotypes to phenotypes in the human gut microbiome.
The microbiota is vital for the development of the immune system and homeostasis. Changes in microbial composition and function, termed dysbiosis, in the respiratory tract and the gut have recently ...been linked to alterations in immune responses and to disease development in the lungs. In this Opinion article, we review the microbial species that are usually found in healthy gastrointestinal and respiratory tracts, their dysbiosis in disease and interactions with the gut-lung axis. Although the gut-lung axis is only beginning to be understood, emerging evidence indicates that there is potential for manipulation of the gut microbiota in the treatment of lung diseases.
Land plants associate with a root microbiota distinct from the complex microbial community present in surrounding soil. The microbiota colonizing the rhizosphere (immediately surrounding the root) ...and the endophytic compartment (within the root) contribute to plant growth, productivity, carbon sequestration and phytoremediation. Colonization of the root occurs despite a sophisticated plant immune system, suggesting finely tuned discrimination of mutualists and commensals from pathogens. Genetic principles governing the derivation of host-specific endophyte communities from soil communities are poorly understood. Here we report the pyrosequencing of the bacterial 16S ribosomal RNA gene of more than 600 Arabidopsis thaliana plants to test the hypotheses that the root rhizosphere and endophytic compartment microbiota of plants grown under controlled conditions in natural soils are sufficiently dependent on the host to remain consistent across different soil types and developmental stages, and sufficiently dependent on host genotype to vary between inbred Arabidopsis accessions. We describe different bacterial communities in two geochemically distinct bulk soils and in rhizosphere and endophytic compartments prepared from roots grown in these soils. The communities in each compartment are strongly influenced by soil type. Endophytic compartments from both soils feature overlapping, low-complexity communities that are markedly enriched in Actinobacteria and specific families from other phyla, notably Proteobacteria. Some bacteria vary quantitatively between plants of different developmental stage and genotype. Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plant-microbe interactions derived from complex soil communities.
The origin of oxygenic photosynthesis in Cyanobacteria led to the rise of oxygen on Earth ~2.3 billion years ago, profoundly altering the course of evolution by facilitating the development of ...aerobic respiration and complex multicellular life. Here we report the genomes of 41 uncultured organisms related to the photosynthetic Cyanobacteria (class Oxyphotobacteria), including members of the class Melainabacteria and a new class of Cyanobacteria (class Sericytochromatia) that is basal to the Melainabacteria and Oxyphotobacteria. All members of the Melainabacteria and Sericytochromatia lack photosynthetic machinery, indicating that phototrophy was not an ancestral feature of the Cyanobacteria and that Oxyphotobacteria acquired the genes for photosynthesis relatively late in cyanobacterial evolution. We show that all three classes independently acquired aerobic respiratory complexes, supporting the hypothesis that aerobic respiration evolved after oxygenic photosynthesis.
Infection with SARS-CoV-2, the etiologic agent of the ongoing COVID-19 pandemic, is accompanied by the shedding of the virus in stool. Therefore, the quantification of SARS-CoV-2 in wastewater ...affords the ability to monitor the prevalence of infections among the population via wastewater-based epidemiology (WBE). In the current work, SARS-CoV-2 RNA was concentrated from wastewater in a catchment in Australia and viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) resulting in two positive detections within a six day period from the same wastewater treatment plant (WWTP). The estimated viral RNA copy numbers observed in the wastewater were then used to estimate the number of infected individuals in the catchment via Monte Carlo simulation. Given the uncertainty and variation in the input parameters, the model estimated a median range of 171 to 1,090 infected persons in the catchment, which is in reasonable agreement with clinical observations. This work highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities. The work also draws attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.
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•First study that reports the detection of SARS-CoV-2 in wastewater in Australia.•The presence of SARS-CoV-2 was confirmed by sequencing.•A median range of 171 to 1090 infected persons was identified in the catchment.•Further methodological and molecular assay validation will be required.
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