DiGAlign: Versatile and Interactive Visualization of Sequence Alignment for Comparative Genomics.

With the explosion of available genomic information, comparative genomics has become a central approach to understanding microbial ecology and evolution. We developed DiGAlign (https://www.genome.jp/digalign/), a web server that provides versatile functionality for comparative genomics with an intuitive interface. It allows the user to perform the highly customizable visualization of a synteny map by simply uploading nucleotide sequences of interest, ranging from a specific region to the whole genome landscape of microorganisms and viruses. DiGAlign will serve a wide range of biological researchers, particularly experimental biologists, with multifaceted features that allow the rapid characterization of genomic sequences of interest and the generation of a publication-ready figure.

Metagenomic analysis of ecological niche overlap and community collapse in microbiome dynamics.

Species utilizing the same resources often fail to coexist for extended periods of time. Such competitive exclusion mechanisms potentially underly microbiome dynamics, causing breakdowns of communities composed of species with similar genetic backgrounds of resource utilization. Although genes responsible for competitive exclusion among a small number of species have been investigated in pioneering studies, it remains a major challenge to integrate genomics and ecology for understanding stable coexistence in species-rich communities. Here, we examine whether community-scale analyses of functional gene redundancy can provide a useful platform for interpreting and predicting collapse of bacterial communities. Through 110-day time-series of experimental microbiome dynamics, we analyzed the metagenome-assembled genomes of co-occurring bacterial species. We then inferred ecological niche space based on the multivariate analysis of the genome compositions. The analysis allowed us to evaluate potential shifts in the level of niche overlap between species through time. We hypothesized that community-scale pressure of competitive exclusion could be evaluated by quantifying overlap of genetically determined resource-use profiles (metabolic pathway profiles) among coexisting species. We found that the degree of community compositional changes observed in the experimental microbiome was correlated with the magnitude of gene-repertoire overlaps among bacterial species, although the causation between the two variables deserves future extensive research. The metagenome-based analysis of genetic potential for competitive exclusion will help us forecast major events in microbiome dynamics such as sudden community collapse (i.e., dysbiosis).

A 1.5-Mb continuous endogenous viral region in the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Most fungal viruses are RNA viruses, and no double-stranded DNA virus that infects fungi is known to date. A recent study detected DNA polymerase genes that originated from large dsDNA viruses in the genomes of basal fungi, suggestive of the existence of dsDNA viruses capable of infecting fungi. In this study, we searched for viral infection signatures in chromosome-level genome assemblies of the arbuscular mycorrhizal fungus Rhizophagus irregularis. We identified a continuous 1.5-Mb putative viral region on a chromosome in R. irregularis strain 4401. Phylogenetic analyses revealed that the viral region is related to viruses in the family Asfarviridae of the phylum Nucleocytoviricota. This viral region was absent in the genomes of four other R. irregularis strains and had fewer signals of fungal transposable elements than the other genomic regions, suggesting a recent and single insertion of a large dsDNA viral genome in the genome of this fungal strain. We also incidentally identified viral-like sequences in the genome assembly of the sea slug Elysia marginata that are evolutionally close to the 1.5-Mb putative viral region. In conclusion, our findings provide strong evidence of the recent infection of the fungus by a dsDNA virus.

A rapid genome-wide analysis of isolated giant viruses using MinION sequencing.

Following the discovery of Acanthamoeba polyphaga mimivirus, diverse giant viruses have been isolated. However, only a small fraction of these isolates have been completely sequenced, limiting our understanding of the genomic diversity of giant viruses. MinION is a portable and low-cost long-read sequencer that can be readily used in a laboratory. Although MinION provides highly error-prone reads that require correction through additional short-read sequencing, recent studies assembled high-quality microbial genomes only using MinION sequencing. Here, we evaluated the accuracy of MinION-only genome assemblies for giant viruses by re-sequencing a prototype marseillevirus. Assembled genomes presented over 99.98% identity to the reference genome with a few gaps, demonstrating a high accuracy of the MinION-only assembly. As a proof of concept, we de novo assembled five newly isolated viruses. Average nucleotide identities to their closest known relatives suggest that the isolates represent new species of marseillevirus, pithovirus and mimivirus. The assembly of subsampled reads demonstrated that their taxonomy and genomic composition could be analysed at the 50× sequencing coverage. We also identified a pithovirus gene whose homologues were detected only in metagenome-derived relatives. Collectively, we propose that MinION-only assembly is an effective approach to rapidly perform a genome-wide analysis of isolated giant viruses.

Predictive Accuracy of the ROX Index for Re-Intubation in Mechanically Ventilated Patients With COVID-19.

BACKGROUND: Several studies have suggested that high-flow nasal cannula (HFNC) is useful for respiratory support after extubation in subjects with COVID-19 pneumonia, whereas 18% subsequently needed to undergo re-intubation. This study aimed to evaluate whether the breathing frequency (f)-ratio of oxygen saturation (ROX) index, which has been shown to be useful for predicting future intubation, is also useful for re-intubation in subjects with COVID-19. METHODS: We retrospectively analyzed mechanically ventilated subjects with COVID-19 who underwent HFNC therapy after extubation at 4 participating hospitals between January 2020-May 2022. We evaluated the predictive accuracy of ROX at 0, 1, and 2 h for re-intubation until ICU discharge and compared the area under the receiver operating characteristic (ROC) curve of the ROX index with those of f and SpO2 /FIO2 . RESULTS: Among the 248 subjects with COVID-19 pneumonia, 44 who underwent HFNC therapy after extubation were included. A total of 32 subjects without re-intubation were classified into the HFNC success group, and 12 with re-intubation were classified into the failure group. The overall trend that the area under the ROC curve of the ROX index was greater than that of the f and SaO2 /FIO2 was observed, although there was no statistical significance at any time point. The ROX index at 0 h, at the cutoff point of < 7.44, showed a sensitivity and specificity of 0.42 and 0.97, respectively. A trend of positive correlation between the time until re-intubation and ROX index at each time point was observed. CONCLUSIONS: The ROX index in the early phase of HFNC therapy after extubation was useful for predicting re-intubation with high accuracy in mechanically ventilated subjects with COVID-19. Close observation for patients with < 7.44 ROX index just after extubation may be warranted because of their high risk for re-intubation.

Facilitative interaction networks in experimental microbial community dynamics.

Facilitative interactions between microbial species are ubiquitous in various types of ecosystems on the Earth. Therefore, inferring how entangled webs of interspecific interactions shift through time in microbial ecosystems is an essential step for understanding ecological processes driving microbiome dynamics. By compiling shotgun metagenomic sequencing data of an experimental microbial community, we examined how the architectural features of facilitative interaction networks could change through time. A metabolic modeling approach for estimating dependence between microbial genomes (species) allowed us to infer the network structure of potential facilitative interactions at 13 time points through the 110-day monitoring of experimental microbiomes. We then found that positive feedback loops, which were theoretically predicted to promote cascade breakdown of ecological communities, existed within the inferred networks of metabolic interactions prior to the drastic community-compositional shift observed in the microbiome time-series. We further applied "directed-graph" analyses to pinpoint potential keystone species located at the "upper stream" positions of such feedback loops. These analyses on facilitative interactions will help us understand key mechanisms causing catastrophic shifts in microbial community structure.

A global synthesis of high-resolution stable isotope data from benthic foraminifera of the last deglaciation.

We present the first version of the Ocean Circulation and Carbon Cycling (OC3) working group database, of oxygen and carbon stable isotope ratios from benthic foraminifera in deep ocean sediment cores from the Last Glacial Maximum (LGM, 23-19 ky) to the Holocene (<10 ky) with a particular focus on the early last deglaciation (19-15 ky BP). It includes 287 globally distributed coring sites, with metadata, isotopic and chronostratigraphic information, and age models. A quality check was performed for all data and age models, and sites with at least millennial resolution were preferred. Deep water mass structure as well as differences between the early deglaciation and LGM are captured by the data, even though its coverage is still sparse in many regions. We find high correlations among time series calculated with different age models at sites that allow such analysis. The database provides a useful dynamical approach to map physical and biogeochemical changes of the ocean throughout the last deglaciation.

Characterization of a bloom-associated alphaproteobacterial lineage, 'Candidatus Phycosocius': insights into freshwater algal-bacterial interactions.

Marine bacterial lineages associated with algal blooms, such as the Roseobacter clade, have been well characterized in ecological and genomic contexts, yet such lineages have rarely been explored in freshwater blooms. This study performed phenotypic and genomic analyses of an alphaproteobacterial lineage 'Candidatus Phycosocius' (denoted the CaP clade), one of the few lineages ubiquitously associated with freshwater algal blooms, and described a novel species: 'Ca. Phycosocius spiralis.' Phylogenomic analyses indicated that the CaP clade is a deeply branching lineage in the Caulobacterales. Pangenome analyses revealed characteristic features of the CaP clade: aerobic anoxygenic photosynthesis and essential vitamin B auxotrophy. Genome size varies widely among members of the CaP clade (2.5-3.7 Mb), likely a result of independent genome reductions at each lineage. This includes a loss of tight adherence pilus genes (tad) in 'Ca. P. spiralis' that may reflect its adoption of a unique spiral cell shape and corkscrew-like burrowing activity at the algal surface. Notably, quorum sensing (QS) proteins showed incongruent phylogenies, suggesting that horizontal transfers of QS genes and QS-involved interactions with specific algal partners might drive CaP clade diversification. This study elucidates the ecophysiology and evolution of proteobacteria associated with freshwater algal blooms.

A Fast and Easy Method to Co-extract DNA and RNA from an Environmental Microbial Sample.

We herein propose a fast and easy DNA and RNA co-extraction method for environmental microbial samples. It combines bead beating and phenol-chloroform phase separation followed by the separation and purification of DNA and RNA using the Qiagen AllPrep DNA/RNA mini kit. With a handling time of ~3 h, our method simultaneously extracted high-quality DNA (peak size >10-15| |kb) and RNA (RNA integrity number >6) from lake bacterioplankton filtered samples. The method is also applicable to low-biomass samples (expected DNA or RNA yield <50| |ng) and eukaryotic microbial samples, providing an easy option for more versatile eco-genomic applications.

Long-Read-Resolved, Ecosystem-Wide Exploration of Nucleotide and Structural Microdiversity of Lake Bacterioplankton Genomes.

Reconstruction of metagenome-assembled genomes (MAGs) has become a fundamental approach in microbial ecology. However, a MAG is hardly complete and overlooks genomic microdiversity because metagenomic assembly fails to resolve microvariants among closely related genotypes. Aiming at understanding the universal factors that drive or constrain prokaryotic genome diversification, we performed an ecosystem-wide high-resolution metagenomic exploration of microdiversity by combining spatiotemporal (2 depths × 12 months) sampling from a pelagic freshwater system, high-quality MAG reconstruction using long- and short-read metagenomic sequences, and profiling of single nucleotide variants (SNVs) and structural variants (SVs) through mapping of short and long reads to the MAGs, respectively. We reconstructed 575 MAGs, including 29 circular assemblies, providing high-quality reference genomes of freshwater bacterioplankton. Read mapping against these MAGs identified 100 to 101,781 SNVs/Mb and 0 to 305 insertions, 0 to 467 deletions, 0 to 41 duplications, and 0 to 6 inversions for each MAG. Nonsynonymous SNVs were accumulated in genes potentially involved in cell surface structural modification to evade phage recognition. Most (80.2%) deletions overlapped with a gene coding region, and genes of prokaryotic defense systems were most frequently (>8% of the genes) overlapped with a deletion. Some such deletions exhibited a monthly shift in their allele frequency, suggesting a rapid turnover of genotypes in response to phage predation. MAGs with extremely low microdiversity were either rare or opportunistic bloomers, suggesting that population persistency is key to their genomic diversification. The results concluded that prokaryotic genomic diversification is driven primarily by viral load and constrained by a population bottleneck. IMPORTANCE Identifying intraspecies genomic diversity (microdiversity) is crucial to understanding microbial ecology and evolution. However, microdiversity among environmental assemblages is not well investigated, because most microbes are difficult to culture. In this study, we performed cultivation-independent exploration of bacterial genomic microdiversity in a lake ecosystem using a combination of short- and long-read metagenomic analyses. The results revealed the broad spectrum of genomic microdiversity among the diverse bacterial species in the ecosystem, which has been overlooked by conventional approaches. Our ecosystem-wide exploration further allowed comparative analysis among the genomes and genes and revealed factors behind microbial genomic diversification, namely, that diversification is driven primarily by resistance against viral infection and constrained by the population size.

Ecogenomics sheds light on diverse lifestyle strategies in freshwater CPR.

BACKGROUND: The increased use of metagenomics and single-cell genomics led to the discovery of organisms from phyla with no cultivated representatives and proposed new microbial lineages such as the candidate phyla radiation (CPR or Patescibacteria). These bacteria have peculiar ribosomal structures, reduced metabolic capacities, small genome, and cell sizes, and a general host-associated lifestyle was proposed for the radiation. So far, most CPR genomes were obtained from groundwaters; however, their diversity, abundance, and role in surface freshwaters is largely unexplored. Here, we attempt to close these knowledge gaps by deep metagenomic sequencing of 119 samples of 17 different freshwater lakes located in Europe and Asia. Moreover, we applied Fluorescence in situ Hybridization followed by Catalyzed Reporter Deposition (CARD-FISH) for a first visualization of distinct CPR lineages in freshwater samples. RESULTS: A total of 174 dereplicated metagenome-assembled genomes (MAGs) of diverse CPR lineages were recovered from the investigated lakes, with a higher prevalence from hypolimnion samples (162 MAGs). They have reduced genomes (median size 1 Mbp) and were generally found in low abundances (0.02-14.36 coverage/Gb) and with estimated slow replication rates. The analysis of genomic traits and CARD-FISH results showed that the radiation is an eclectic group in terms of metabolic capabilities and potential lifestyles, ranging from what appear to be free-living lineages to host- or particle-associated groups. Although some complexes of the electron transport chain were present in the CPR MAGs, together with ion-pumping rhodopsins and heliorhodopsins, we believe that they most probably adopt a fermentative metabolism. Terminal oxidases might function in O2 scavenging, while heliorhodopsins could be involved in mitigation against oxidative stress. CONCLUSIONS: A high diversity of CPR MAGs was recovered, and distinct CPR lineages did not seem to be limited to lakes with specific trophic states. Their reduced metabolic capacities resemble the ones described for genomes in groundwater and animal-associated samples, apart from Gracilibacteria that possesses more complete metabolic pathways. Even though this radiation is mostly host-associated, we also observed organisms from different clades (ABY1, Paceibacteria, Saccharimonadia) that appear to be unattached to any other organisms or were associated with 'lake snow' particles (ABY1, Gracilibacteria), suggesting a broad range of potential life-strategies in this phylum. Video Abstract.

Electrodialytic Enrichment and Matrix Conversion for the Determination of Trace Metals in Ultra-Pure Water.

The presence of trace contaminants in ultra-pure water (UPW) used in fabrication process can greatly affect the yield and quality of industrial products. In the present study, the electrodialytic enrichment of metal cations as a means of continuously monitoring the UPW quality was studied. A newly designed electrodialytic enrichment device (EED) was used to quantitatively transfer metal ions from samples to dilute nitric acid, which was then directly introduced into an inductively coupled plasma-mass spectrometry (ICP-MS) instrument. This process could be performed without contamination of the sample, and the enrichment factor was solely dependent on the flow rate ratio of the sample and acceptor solutions. The transference of analytes into the acidic solution improved the responsivity of the ICP-MS analysis, especially at low concentrations of less than 1 µg/L. Blank solutions to support the analysis of UPW could be produced using the EED effluent, from which metal ions were quantitatively removed. In addition, calibration curves with concentration ranges of several nanograms per liter were obtained by preparing standards using a dynamic gravimetric method while employing a single bottle and continuous mass monitoring to avoid any contamination from the volumetric flasks. The sensitivities associated with the ICP-MS analysis of a number of trace metal ions were improved by one or two orders of magnitude. The data show that the present EED is able to continuously produce enriched analyte solutions to allow the ongoing monitoring of UPW quality.

Long-read metagenomics using PromethION uncovers oral bacteriophages and their interaction with host bacteria.

Bacteriophages (phages), or bacterial viruses, are very diverse and highly abundant worldwide, including as a part of the human microbiomes. Although a few metagenomic studies have focused on oral phages, they relied on short-read sequencing. Here, we conduct a long-read metagenomic study of human saliva using PromethION. Our analyses, which integrate both PromethION and HiSeq data of >30 Gb per sample with low human DNA contamination, identify hundreds of viral contigs; 0-43.8% and 12.5-56.3% of the confidently predicted phages and prophages, respectively, do not cluster with those reported previously. Our analyses demonstrate enhanced scaffolding, and the ability to place a prophage in its host genomic context and enable its taxonomic classification. Our analyses also identify a Streptococcus phage/prophage group and nine jumbo phages/prophages. 86% of the phage/prophage group and 67% of the jumbo phages/prophages contain remote homologs of antimicrobial resistance genes. Pan-genome analysis of the phages/prophages reveals remarkable diversity, identifying 0.3% and 86.4% of the genes as core and singletons, respectively. Furthermore, our study suggests that oral phages present in human saliva are under selective pressure to escape CRISPR immunity. Our study demonstrates the power of long-read metagenomics utilizing PromethION in uncovering bacteriophages and their interaction with host bacteria.

Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing.

BACKGROUND: Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. RESULTS: Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7-101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. CONCLUSIONS: Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future. Video abstract.

Impact of atrial fibrillation on the risk of ischemic stroke in patients on hemodialysis: BOREAS-HD3 Study.

BACKGROUND: Atrial fibrillation (AF) is an established risk factor for ischemic stroke in a general population. However, its impact in patients on hemodialysis (HD), a group with a high risk for stroke, is still controversial. Here we examined this issue in a Japanese cohort. METHODS: This study was designed as a multicenter cohort study. HD patients (n = 1,067) were enrolled from 22 institutes in January 2009 and followed up for 3 years. Patients with missing data (n = 196) or kidney transplantation (n = 4) were excluded, and 867 patients contributed to the analysis of the risk of new-onset of ischemic stroke. RESULTS: At baseline, AF was observed in 123 patients (14.2%, AF group) and not in the others (n = 744: 85.8%, non-AF group). During a follow-up period of 31.3 months, the cumulative incidence rate for ischemic stroke was significantly higher in the AF group than in the non-AF group (6.5% vs. 2.9%, p < 0.05). In Cox regression analysis, AF was a significant independent risk factor for new-onset of ischemic stroke after adjustment for age, sex, prior history of ischemic stroke, use of warfarin, dialysis vintage, comorbidity of diabetic nephropathy, and interdialytic weight gain (hazard ratio 2.17-2.68). CONCLUSION: Present analyses using comprehensive adjustment for multiple confounders, including prior history of ischemic stroke, indicated that AF independently increases the risk of new-onset of ischemic stroke by more than twofold in Japanese HD patients.

Widespread Dominance of Kinetoplastids and Unexpected Presence of Diplonemids in Deep Freshwater Lakes.

Kinetoplastid flagellates are generally abundant in the deep sea and recently they were even found to be dominant in the hypolimnion of a deep freshwater lake. Therefore, to understand the distribution of kinetoplastids in deep freshwater lakes, we have collected vertical samples from five lakes in Japan. The abundance of kinetoplastids was enumerated by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization, and the diversity was determined by 18S amplicon sequencing using universal eukaryote and kinetoplastid-specific primers. Kinetoplastids were abundant in the deep waters of all the lakes, contributing up to 53.6% of total nanoeukaryotes. Despite this significant contribution, kinetoplastids remain undetected by amplicon sequencing using universal primers that are widely used in eukaryotic diversity studies. However, they were detected with specific primers, and the communities were characterized by both ubiquitous and lake-specific unique OTUs. Oligotyping of a ubiquitous and dominant OTU revealed the presence of lake-specific sequence types (oligotypes). Remarkably, we also detected diplonemids (a sister group of kinetoplastids and considered to be specific in the marine habitat) using kinetoplastid-specific primers, showing their presence in freshwaters. Underestimation of kinetoplastids and diplonemids using universal primers indicates that euglenozoan flagellates are overlooked in diversity studies worldwide. The present study highlighted the importance of kinetoplastids in the hypolimnion of deep lakes, thereby indicating their role in material cycling in deep waters.

Genome-resolved viral and cellular metagenomes revealed potential key virus-host interactions in a deep freshwater lake.

Metagenomics has dramatically expanded the known virosphere, but freshwater viral diversity and their ecological interaction with hosts remain poorly understood. Here, we conducted a metagenomic exploration of planktonic dsDNA prokaryotic viruses by sequencing both virion (<0.22 µm) and cellular (0.22-5.0 µm) fractions collected spatiotemporally from a deep freshwater lake (Lake Biwa, Japan). This simultaneously reconstructed 183 complete (i.e., circular) viral genomes and 57 bacterioplankton metagenome-assembled genomes. Analysis of metagenomic read coverage revealed vertical partitioning of the viral community analogous to the vertically stratified bacterioplankton community. The hypolimnetic community was generally stable during stratification, but occasionally shifted abruptly, presumably due to lysogenic induction. Genes involved in assimilatory sulfate reduction were encoded in 20 (10.9%) viral genomes, including those of dominant viruses, and may aid viral propagation in sulfur-limited freshwater systems. Hosts were predicted for 40 (21.9%) viral genomes, encompassing 10 phyla (or classes of Proteobacteria) including ubiquitous freshwater bacterioplankton lineages (e.g., Ca. Fonsibacter and Ca. Nitrosoarchaeum). Comparison with viral genomes derived from published metagenomes revealed viral phylogeographic connectivity in geographically isolated habitats. Notably, analogous to their hosts, actinobacterial viruses were among the most diverse, ubiquitous and abundant viral groups in freshwater systems, with potential high lytic activity in surface waters.

Use of plant hormones to activate silent polyketide biosynthetic pathways in Arthrinium sacchari, a fungus isolated from a spider.

Plant hormones were evaluated for their ability to activate fungal secondary metabolite production. Three synthetic cytokinins, kinetin, 6-benzylaminopurine, and forchlorfenuron, showed remarkable enhancement of the production of aromatic polyketides derived from emodin in a fungus, Arthrinium sacchari, and allowed us to isolate a new polyketide. Furthermore, we firstly demonstrated the potential of plant hormones to activate a wide range of fungal secondary metabolite production processes.