Metagenomic analysis reveals altered gut virome and diagnostic potential in pancreatic cancer.

Pancreatic cancer (PC) is a highly aggressive malignancy with a poor prognosis, making early diagnosis crucial for improving patient outcomes. While the gut microbiome, including bacteria and viruses, is believed to be essential in cancer pathogenicity, the potential contribution of the gut virome to PC remains largely unexplored. In this study, we conducted a comparative analysis of the gut viral compositional and functional profiles between PC patients and healthy controls, based on fecal metagenomes from two publicly available data sets comprising a total of 101 patients and 82 healthy controls. Our results revealed a decreasing trend in the gut virome diversity of PC patients with disease severity. We identified significant alterations in the overall viral structure of PC patients, with a meta-analysis revealing 219 viral operational taxonomic units (vOTUs) showing significant differences in relative abundance between patients and healthy controls. Among these, 65 vOTUs were enriched in PC patients, and 154 were reduced. Host prediction revealed that PC-enriched vOTUs preferentially infected bacterial members of Veillonellaceae, Enterobacteriaceae, Fusobacteriaceae, and Streptococcaceae, while PC-reduced vOTUs were more likely to infect Ruminococcaceae, Lachnospiraceae, Clostridiaceae, Oscillospiraceae, and Peptostreptococcaceae. Furthermore, we constructed random forest models based on the PC-associated vOTUs, achieving an optimal average area under the curve (AUC) of up to 0.879 for distinguishing patients from controls. Through additional 10 public cohorts, we demonstrated the reproducibility and high specificity of these viral signatures. Our study suggests that the gut virome may play a role in PC development and could serve as a promising target for PC diagnosis and therapeutic intervention. Future studies should further explore the underlying mechanisms of gut virus-bacteria interactions and validate the diagnostic models in larger and more diverse populations.

Testing latent classes in gut microbiome data using generalized Poisson regression models.

Human microbiome research has gained increasing importance due to its critical roles in comprehending human health and disease. Within the realm of microbiome research, the data generated often involves operational taxonomic unit counts, which can frequently present challenges such as over-dispersion and zero-inflation. To address dispersion-related concerns, the generalized Poisson model offers a flexible solution, effectively handling data characterized by over-dispersion, equi-dispersion, and under-dispersion. Furthermore, the realm of zero-inflated generalized Poisson models provides a strategic avenue to simultaneously tackle both over-dispersion and zero-inflation. The phenomenon of zero-inflation frequently stems from the heterogeneous nature of study populations. It emerges when specific microbial taxa fail to thrive in the microbial community of certain subjects, consequently resulting in a consistent count of zeros for these individuals. This subset of subjects represents a latent class, where their zeros originate from the genuine absence of the microbial taxa. In this paper, we introduce a novel testing methodology designed to uncover such latent classes within generalized Poisson regression models. We establish a closed-form test statistic and deduce its asymptotic distribution based on estimating equations. To assess its efficacy, we conduct an extensive array of simulation studies, and further apply the test to detect latent classes in human gut microbiome data from the Bogalusa Heart Study.

Fungal community and toxigenic taxa in chestnut fruits in postharvest conditioning process and storage.

BACKGROUND: Chestnut fruit quality is affected by fungal contamination. The study of the patterns of contamination in the postharvest is crucial to individuate the critical phases and propose solutions. To understand how fungal colonization varies on fruits, the composition of mycobiota was investigated in postharvest handling and in between tissues (shell and kernel). RESULTS: Fungal sequences were clustered into 308 operational taxonomic units (OTUs). Biodiversity was higher in shell than kernel tissues. Results evidenced the risk of new contamination in specific phases such as the 'cold bath' and storage. Genera known as mycotoxin producers were detected in all phases. Specifically, 47 OTUs belonging to Penicillium, eight to Fusarium and two to Aspergillus genera were identified. While Fusarium spp. was sensitive to 'warm bath' phase, Penicillium spp. was largely insensitive and accumulated in storage conditions. Surprisingly, Aspergillus spp. was poorly represented. Aflatoxin, ochratoxin A, fumonisins and T-2/HT-2 detection was performed for shell and kernel, and process phases. Higher contamination was observed on shell than in kernel samples. While aflatoxins were within the European Union (EU) limits for dry fruits, Ochratoxin exceeded the EU limits. The present study represents the first report of fumonisins and T-2/HT-2 detection in chestnuts. CONCLUSION: Fungal contamination taxa is high in chestnut fruits following postharvest handling and storage. A parametrization of process phases such as the 'warm bath' is functional to reduce the risk for some taxa. For other spoilage and mycotoxigenic genera strict sanitation procedures of equipment and water must be individuated and implemented to reduce their impact. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Community metabarcoding reveals the relative role of environmental filtering and spatial processes in metacommunity dynamics of soil microarthropods across a mosaic of montane forests.

Disentangling the relative role of environmental filtering and spatial processes in driving metacommunity structure across mountainous regions remains challenging, as the way we quantify spatial connectivity in topographically and environmentally heterogeneous landscapes can influence our perception of which process predominates. More empirical data sets are required to account for taxon- and context-dependency, but relevant research in understudied areas is often compromised by the taxonomic impediment. Here we used haplotype-level community DNA metabarcoding, enabled by stringent filtering of amplicon sequence variants (ASVs), to characterize metacommunity structure of soil microarthropod assemblages across a mosaic of five forest habitats on the Troodos mountain range in Cyprus. We found similar ß diversity patterns at ASV and species (OTU, operational taxonomic unit) levels, which pointed to a primary role of habitat filtering resulting in the existence of largely distinct metacommunities linked to different forest types. Within-habitat turnover was correlated to topoclimatic heterogeneity, again emphasizing the role of environmental filtering. However, when integrating landscape matrix information for the highly fragmented Quercus alnifolia habitat, we also detected a major role of spatial isolation determined by patch connectivity, indicating that stochastic and niche-based processes synergistically govern community assembly. Alpha diversity patterns varied between ASV and OTU levels, with OTU richness decreasing with elevation and ASV richness following a longitudinal gradient, potentially reflecting a decline of genetic diversity eastwards due to historical pressures. Our study demonstrates the utility of haplotype-level community metabarcoding for characterizing metacommunity structure of complex assemblages and improving our understanding of biodiversity dynamics across mountainous landscapes worldwide.

The Diversity Patterns of Rare to Abundant Microbial Eukaryotes Across a Broad Range of Salinities in a Solar Saltern.

Solar salterns are excellent artificial systems for examining species diversity and succession along salinity gradients. Here, the eukaryotic community in surface water of a Korean solar saltern (30 to 380 practical salinity units) was investigated from April 2019 to October 2020 using Illumina sequencing targeting the V4 and V9 regions of 18S rDNA. A total of 926 operational taxonomic units (OTUs) and 1,999 OTUs were obtained with the V4 and V9 regions, respectively. Notably, most of the OTUs were microbial eukaryotes, and the high-abundance groups (> 5% relative abundance (RA), Alveolata, Stramenopila, Archaeplastida, and Opisthokonta) usually accounted for > 90% of the total cumulative read counts and > 80% of all OTUs. Moreover, the high-abundance Alveolata (larger forms) and Stramenopila (smaller forms) groups displayed a significant inverse relationship, probably due to predator-prey interactions. Most of the low-abundance (0.1-5% RA) and rare (< 0.1% RA) groups remained small portion during the field surveys. Taxonomic novelty (at < 90% sequence identity) was high in the Amoebozoa, Cryptista, Haptista, Rhizaria, and Stramenopila groups (69.8% of all novel OTUs), suggesting the presence of a large number of hidden species in hypersaline environments. Remarkably, the high-abundance groups had little overlap with the other groups, implying the weakness of rare-to-prevalent community dynamics. The low-abundance Discoba group alone temporarily became the high-abundance group, suggesting that it is an opportunistic group. Overall, the composition and diversity of the eukaryotic community in hypersaline environments may be persistently stabilized, despite diverse disturbance events.

To denoise or to cluster, that is not the question: optimizing pipelines for COI metabarcoding and metaphylogeography.

BACKGROUND: The recent blooming of metabarcoding applications to biodiversity studies comes with some relevant methodological debates. One such issue concerns the treatment of reads by denoising or by clustering methods, which have been wrongly presented as alternatives. It has also been suggested that denoised sequence variants should replace clusters as the basic unit of metabarcoding analyses, missing the fact that sequence clusters are a proxy for species-level entities, the basic unit in biodiversity studies. We argue here that methods developed and tested for ribosomal markers have been uncritically applied to highly variable markers such as cytochrome oxidase I (COI) without conceptual or operational (e.g., parameter setting) adjustment. COI has a naturally high intraspecies variability that should be assessed and reported, as it is a source of highly valuable information. We contend that denoising and clustering are not alternatives. Rather, they are complementary and both should be used together in COI metabarcoding pipelines. RESULTS: Using a COI dataset from benthic marine communities, we compared two denoising procedures (based on the UNOISE3 and the DADA2 algorithms), set suitable parameters for denoising and clustering, and applied these steps in different orders. Our results indicated that the UNOISE3 algorithm preserved a higher intra-cluster variability. We introduce the program DnoisE to implement the UNOISE3 algorithm taking into account the natural variability (measured as entropy) of each codon position in protein-coding genes.  This correction increased the number of sequences retained by 88%. The order of the steps (denoising and clustering) had little influence on the final outcome. CONCLUSIONS: We highlight the need for combining denoising and clustering, with adequate choice of stringency parameters, in COI metabarcoding. We present a program that uses the coding properties of this marker to improve the denoising step. We recommend researchers to report their results in terms of both denoised sequences (a proxy for haplotypes) and clusters formed (a proxy for species), and to avoid collapsing the sequences of the latter into a single representative. This will allow studies at the cluster (ideally equating species-level diversity) and at the intra-cluster level, and will ease additivity and comparability between studies.

City-Scale Meta-Analysis of Indoor Airborne Microbiota Reveals that Taxonomic and Functional Compositions Vary with Building Types.

Currently, there is a lack of understanding on the variations of the indoor airborne microbiotas of different building types within a city, and how operational taxonomic unit (OTU)- and amplicon sequence variant (ASV)-based analyses of the 16S rRNA gene sequences affect interpretation of the indoor airborne microbiota results. Therefore, in this study, the indoor airborne bacterial microbiotas between commercial buildings, residences, and subways within the same city were compared using both OTU- and ASV-based analytic methods. Our findings suggested that indoor airborne bacterial microbiota compositions were significantly different between building types regardless of the bioinformatics method used. The processes of ecological drift and random dispersal consistently played significant roles in the assembly of the indoor microbiota across building types. Abundant taxa tended to be more centralized in the correlation network of each building type, highlighting their importance. Taxonomic changes between the microbiotas of different building types were also linked to changes in their inferred metabolic function capabilities. Overall, the results imply that customized strategies are necessary to manage indoor airborne bacterial microbiotas for each building type or even within each specific building.

CDSnake: Snakemake pipeline for retrieval of annotated OTUs from paired-end reads using CD-HIT utilities.

BACKGROUND: Illumina paired-end reads are often used for 16S analysis in metagenomic studies. Since DNA fragment size is usually smaller than the sum of lengths of paired reads, reads can be merged for downstream analysis. In spite of development of several tools for merging of paired-end reads, poor quality at the 3' ends within the overlapping region prevents the accurate combining of significant portion of read pairs. Recently CD-HIT-OTU-Miseq was presented as a new approach for 16S analysis using the paired-end reads, it completely avoids the reads merging process due to separate clustering of paired reads. CD-HIT-OTU-Miseq is a set of tools which are supposed to be successively launched by auxiliary shell scripts. This launch mode is not suitable for processing of big amounts of data generated in modern omics experiments. To solve this issue we created CDSnake - Snakemake pipeline utilizing CD-HIT tools for easier consecutive launch of CD-HIT-OTU-Miseq tools for complete processing of paired end reads in metagenomic studies. Usage of pipeline make 16S analysis easier due to one-command launch and helps to yield reproducible results. RESULTS: We benchmarked our pipeline against two commonly used pipelines for OTU retrieval, incorporated into popular workflow for microbiome analysis, QIIME2 - DADA2 and deblur. Three mock datasets having highly overlapping paired-end 2 × 250 bp reads were used for benchmarking - Balanced, HMP, and Extreme. CDSnake outputted less OTUs than DADA2 and deblur. However, on Balanced and HMP datasets number of OTUs outputted by CDSnake was closer to real number of strains which were used for mock community generation, than those outputted by DADA2 and deblur. Though generally slower than other pipelines, CDSnake outputted higher total counts, preserving more information from raw data. Inheriting this properties from original CD-HIT-OTU-MiSeq utilities, CDSnake made their usage handier due to simple scalability, easier automated runs and other Snakemake benefits. CONCLUSIONS: We developed Snakemake pipeline for OTU-MiSeq utilities, which simplified and automated data analysis. Benchmarking showed that this approach is capable to outperform popular tools in certain conditions.

Natrix: a Snakemake-based workflow for processing, clustering, and taxonomically assigning amplicon sequencing reads.

BACKGROUND: Sequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires efficient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an efficient workflow management system. RESULTS: We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub ( https://github.com/MW55/Natrix ) or as a Docker container on DockerHub ( https://hub.docker.com/r/mw55/natrix ). CONCLUSION: Natrix is a user-friendly and highly extensible workflow for processing Illumina amplicon data.

Integrative description of bisexual Paramacrobiotus experimentalis sp. nov. (Macrobiotidae) from republic of Madagascar (Africa) with microbiome analysis.

In a moss samples collected on Madagascar two populations of Paramacrobiotus experimentalis sp. nov. were found. Paramacrobiotus experimentalis sp. nov. with the presence of a microplacoid and areolatus type of eggs is similar to Pam. danielae, Pam. garynahi, Pam. hapukuensis, Pam. peteri, Pam. rioplatensis and Pam. savai, but it differs from them by some morphological and morphometric characters of the eggs. The p-distance between two COI haplotypes of Pam. experimentalis sp. nov. was 0.17%. In turn, the ranges of uncorrected genetic p-distances of all Paramacrobiotus species available in GenBank was from 18.27% (for Pam. lachowskae) to 25.26% (for Pam. arduus) with an average distance of 20.67%. We also found that Pam. experimentalis sp. nov. is bisexual. This observation was congruent on three levels: (i) morphological - specimen size dimorphism; (ii) structural (primary sexual characteristics) - females have an unpaired ovary while males have an unpaired testis and (iii) molecular - heterozygous and homozygous strains of the ITS-2 marker. Although symbiotic associations of hosts with bacteria (including endosymbiotic bacteria) are common in nature and these interactions exert various effects on the evolution, biology and reproductive ecology of hosts, there is still very little information on the bacterial community associated with tardigrades. To fill this gap and characterise the bacterial community of Pam. experimentalis sp. nov. populations and microbiome of its microhabitat, high throughput sequencing of the V3-V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed. The obtained 16S rRNA gene sequences ranged from 92,665 to 131,163. In total, 135 operational taxonomic units (OTUs) were identified across the rarefied dataset. Overall, both Pam. experimentalis sp. nov. populations were dominated by OTUs ascribed to the phylum Proteobacteria (89-92%) and Firmicutes (6-7%). In the case of samples from tardigrades' laboratory habitat, the most abundant bacterial phylum was Proteobacteria (51-90%) and Bacteroides (9-48%). In all compared microbiome profiles, only 16 of 137 OTUs were shared. We found also significant differences in beta diversity between the partly species-specific microbiome of Pam. experimentalis sp. nov. and its culturing environment. Two OTUs belonging to a putative bacterial endosymbiont were identified - Rickettsiales and Polynucleobacter. We also demonstrated that each bacterial community was rich in genes involved in membrane transport, amino acid metabolism, and carbohydrate metabolism.

Soil fungal taxonomic diversity along an elevation gradient on the semi-arid Xinglong Mountain, Northwest China.

Elevation gradients, often regarded as "natural experiments or laboratories", can be used to study changes in the distribution of microbial diversity related to changes in environmental conditions that typically occur over small geographical scales. We exploited this feature by characterizing fungal composition and diversity along an elevation gradient on Xinglong Mountain, northwest China. For this, we used MiSeq sequencing to obtain fungal sequences and clustered them into operational taxonomic units (OTUs). In total, we obtained 1,203,302 reads, 133,700 on average in each sample of soil collected at three selected elevations (2807, 3046, and 3536 m). The reads were assigned to 2192 OTUs. Inconsistent variations were observed in fungal alpha-diversity in samples from the three elevations. However, Principal Coordinate Analysis based on Bray-Curtis and UniFrac (weighted and unweighted) distance metrics revealed that fungal communities in soil samples from 3046 and 3536 m elevations were most similar. Principal Component Analysis based on relative abundances of shared OTUs confirmed that OTUs in samples from 3536 m elevation were more closely related to OTUs from 3046 m than samples from 2807 m elevation. Ascomycota, Basidiomycota, Glomeromycota, Cercozoa and Chytridiomycota were the most abundant fungal phyla across the elevation gradient. Our study also provides valuable indications of relations between fungal communities and an array of soil chemical properties, and variations in fungal taxonomic diversity across a substantial elevation gradient.

A comparative study of microbial community and dynamics of Asaia in the brown planthopper from susceptible and resistant rice varieties.

BACKGROUND: The brown planthopper (BPH) is likely the most destructive, piercing and sucking monophagous insect pest of rice that causes substantial economic losses to farmers. Although yeast-like symbionts (YLS) and virus transmission have been observed in the BPH, the bacterial population inhabiting the BPH has received minimal research attention. Labelling BPH-associated bacterial species may shed light on BPH biology and the interaction between the BPH and rice to provide novel approaches for the efficient control of this insect pest. RESULTS: We examined RNA-seq results to identify bacterial populations present in different generations of BPHs maintained on susceptible or resistant rice varieties. Overall, 87 operational taxonomic units (OTUs) were determined from the BPH-F0, F6 and F16 generations. These OTUs had Shannon and Simpson index values of 0.37-0.6 and 0.56-1.19, respectively. The evenness values of 0.7-1.00 showed the vastness of the bacterial diversity recovered from the BPH samples. The results showed high species diversity in the BPHs collected from susceptible rice and a high number of members of unclassified bacteria in the BPHs isolated from resistant rice. We noticed that Proteobacteria OTUs were predominant across all samples. Furthermore, PCR data of Asaia species showed variable DNA amplification across the BPH samples collected from susceptible or resistant varieties. The identification of Asaia in BPH eggs and BPH-egg-infected rice revealed its influence on the interaction between the BPH egg and rice. CONCLUSIONS: The BPHs had clear differences in their microbiomes and in their ability to feed on different rice hosts. These variations could have an essential impact on host adaptation and interaction. These results provide a better understanding of the bacterial diversity and interaction of the microbiome of different generations of BPHs. Furthermore, PCR data of Asaia sp. variation across the BPH samples (isolated from different host genotypes selected from the field and laboratory, including BPH eggs and egg-infected rice tissues), suggest that Asaia could be an important member of the insect microbiome involved in adaptation, its interaction with rice and, most importantly, as a paratransgenic tool for insect control.

The relative importance of ecological drivers of arbuscular mycorrhizal fungal distribution varies with taxon phylogenetic resolution.

The phylogenetic depth at which arbuscular mycorrhizal (AM) fungi harbor a coherent ecological niche is unknown, which has consequences for operational taxonomic unit (OTU) delineation from sequence data and the study of their biogeography. We tested how changes in AM fungi community composition across habitats (beta diversity) vary with OTU phylogenetic resolution. We inferred exact sequence variants (ESVs) to resolve phylotypes at resolutions finer than provided by traditional sequence clustering and analyzed beta diversity profiles up to order-level sequence clusters. At the ESV level, we detected the environmental predictors revealed with traditional OTUs or at higher genetic distances. However, the correlation between environmental predictors and community turnover steeply increased at a genetic distance of c. 0.03 substitutions per site. Furthermore, we observed a turnover of either closely or distantly related taxa (respectively at or above 0.03 substitutions per site) along different environmental gradients. This study suggests that different axes of AM fungal ecological niche are conserved at different phylogenetic depths. Delineating AM fungal phylotypes using DNA sequences should screen different phylogenetic resolutions to better elucidate the factors that shape communities and predict the fate of AM symbioses in a changing environment.

Mosquito control actions affect chironomid diversity in temporary wetlands of the Upper Rhine Valley.

The Upper Rhine Valley, a "hotspot of biodiversity" in Germany, has been treated with the biocide Bacillus thuringiensis var. israelensis (Bti) for mosquito control for decades. Previous studies discovered Bti nontarget effects in terms of severe chironomid abundance reductions. In this study, we investigated the impact of Bti on species level and addressed the community composition of the nontarget family Chironomidae by use of community metabarcoding. Chironomid emergence data were collected in three mosquito-control relevant wetland types in the Upper Rhine Valley. For all three sites the chironomid species composition, based on operational taxonomic units (OTUs), was different to varying degrees in the Bti-treated samples versus control samples, ranging from a significant 63% OTU reduction to an OTU replacement. We assumed that predatory chironomids are less prone to Bti than filter feeders, as the latter feed on floating particles leading to direct ingestion of Bti. However, a comparable percentage of predators and filter feeders (63% and 65%, respectively) was reduced in the Bti samples, suggesting that the feeding strategy is not the main driver for Bti sensitivity in chironomids. Finally, our data was compared to a three-year-old data set, indicating possible chironomid community recovery due to species recolonization a few years after the last Bti application. Considering the currently discussed worldwide insect decline we recommend a rethinking of the usage of the biocide Bti, and to prevent its ongoing application especially in nature protection reserves to enhance ecological resilience and to prevent boosting the current biodiversity loss.

A combined approach of 16S rRNA and a functional marker gene, soxB to reveal the diversity of sulphur-oxidising bacteria in thermal springs.

With the advent of new molecular tools, new taxa of sulphur-oxidising bacteria (SOB) in diverse environments are being discovered. However, there is a significant gap of knowledge about the ecology and diversity of SOB in thermal springs. Here, the species diversity and phylogenetic affiliations of SOB were investigated using 16S rRNA and functional gene marker, soxB in thermal springs of Thane district of Maharashtra, India. Most SOB detected by 16S rDNA sequences belong to different operational taxonomic units (OTU's): Firmicutes, α-, ß-, γ-Proteobacteria and Actinobacteria with the dominance of first class. However, the soxB gene clone library sequences had shown affiliation with the ß-, γ- and α-Proteobacteria. ß-Proteobacteria-related sequences were dominant, with 53.3% clones belonging to genus Hydrogenophaga. The thiosulphate oxidation assay carried out for different isolates having distinct identity showed the mean sulphate-sulphur production from 117.86 ± 0.50 to 218.82 ± 2.56 mg SO4-S l-1 after 9 days of incubation. Also, sulphur oxidation by the genus Nitratireductor, Caldimonas, Geobacillus, Paenibacillus, Brevibacillus, Tristrella and Chelatococcus has been reported for the first time that reveals ecological widening over which thiotrophs are distributed.

Autism spectrum disorder is associated with gut microbiota disorder in children.

BACKGROUND: The aim of this study was to evaluate the occurrence and clinical characteristics of autism spectrum disorder (ASD) associated to the stable state of the gut microbiota. METHODS: A total of 9 children with ASD and 6 healthy children used as control were selected and feces samples were collected from all of them. The 16S gene ribosomal RNA sequencing was used to analyze the difference in gut microbiota between healthy control children and ASD patients. RESULTS: The results of 16S sequencing based on operational taxonomic units (OTUs) analysis showed that the ASD group and the healthy control (HC) group had a large difference in the abundance of microbiota at the level of family, genus and species. The abundance of Bacteroidales and Selenomonadales was significantly lower in the ASD group than in the HC group (p = 0.0110 and p = 0.0076, respectively). The abundance of Ruminococcaceae in the ASD group was higher than that in the HC group (p = 0.0285), while the amount of Prevotellaceae was significantly lower in the ASD group than in the HC group (p = 0.0111). The Tax4Fun analysis based on Kyoto Encyclopaedia of Genes and Genomes (KEGG) data indicated differentially expressed functional pathway between the ASD group and healthy control group associated to the nervous system, environmental information processing and cellular processing. CONCLUSIONS: The abundance of gut microbiota in the ASD group is different from that in the healthy control children. These differences affect the biological function of the host. These results suggest that a disorder in the gut microbiota may be associated, at least in part, with ASD in children.

GeFaST: An improved method for OTU assignment by generalising Swarm's fastidious clustering approach.

BACKGROUND: Massive genomic data sets from high-throughput sequencing allow for new insights into complex biological systems such as microbial communities. Analyses of their diversity and structure are typically preceded by clustering millions of 16S rRNA gene sequences into OTUs. Swarm introduced a new clustering strategy which addresses important conceptual and performance issues of the popular de novo clustering approach. However, some parts of the new strategy, e.g. the fastidious option for increased clustering quality, come with their own restrictions. RESULTS: In this paper, we present the new exact, alignment-based de novo clustering tool GeFaST, which implements a generalisation of Swarm's fastidious clustering. Our tool extends the fastidious option to arbitrary clustering thresholds and allows to adjust its greediness. GeFaST was evaluated on mock-community and natural data and achieved higher clustering quality and performance for small to medium clustering thresholds compared to Swarm and other de novo tools. Clustering with GeFaST was between 6 and 197 times as fast as with Swarm, while the latter required up to 38% less memory for non-fastidious clustering but at least three times as much memory for fastidious clustering. CONCLUSIONS: GeFaST extends the scope of Swarm's clustering strategy by generalising its fastidious option, thereby allowing for gains in clustering quality, and by increasing its performance (especially in the fastidious case). Our evaluations showed that GeFaST has the potential to leverage the use of the (fastidious) clustering strategy for higher thresholds and on larger data sets.

Iron supplementation has minor effects on gut microbiota composition in overweight and obese women in early pregnancy.

Fe is an essential nutrient for many bacteria, and Fe supplementation has been reported to affect the composition of the gut microbiota in both Fe-deficient and Fe-replete individuals outside pregnancy. This study examined whether the dose of Fe in pregnancy multivitamin supplements affects the overall composition of the gut microbiota in overweight and obese pregnant women in early pregnancy. Women participating in the SPRING study with a faecal sample obtained at 16 weeks' gestation were included in this substudy. For each subject, the brand of multivitamin used was recorded. Faecal microbiome composition was assessed by 16S rRNA sequencing and analysed with the QIIME software suite. Dietary intake of Fe was assessed using a FFQ at 16 weeks' gestation. Women were grouped as receiving low (<60 mg/d, n 94) or high (≥60 mg/d; n 65) Fe supplementation. The median supplementary Fe intake in the low group was 10 (interquartile range (IQR) 5-10) v. 60 (IQR 60-60) mg/d in the high group (P<0·001). Dietary Fe intake did not differ between the groups (10·0 (IQR 7·4-13·3) v. 9·8 (IQR 8·2-13·2) mg/d). Fe supplementation did not significantly affect the composition of the faecal microbiome at any taxonomic level. Network analysis showed that the gut microbiota in the low Fe supplementation group had a higher predominance of SCFA producers. Pregnancy multivitamin Fe content has a minor effect on the overall composition of the gut microbiota of overweight and obese pregnant women at 16 weeks' gestation.

Ethanolamine enhances intestinal functions by altering gut microbiome and mucosal anti-stress capacity in weaned rats.

Ethanolamine (Etn) contained in milk is the base constituent of phosphatidylethanolamine and is required for the proliferation of intestinal epithelial cells and bacteria, which is important for maintenance of the gut microbiome and intestinal development. The present study investigated the effect of Etn on intestinal function and microbiome using 21-d-old Sprague-Dawley rats treated with 0, 250, 500 and 1000 µm Etn in drinking water for 2 weeks immediately after weaning. Growth performance, intestinal morphology, antioxidant capacity and mucosal immunity, as well as gut microbiota community composition, were evaluated. Metagenomic prediction and metabolic phenotype analysis based on 16S RNA sequencing were also carried out to assess changes in metabolic functions. We found that weaned rats administered 500 µm Etn enhanced mucosal antioxidant capacity, as evidenced by higher superoxide dismutase and glutathione peroxidase levels in the jejunum (P<0·05) compared with those in the control group. Predominant microbes including Bacteroidetes, Proteobacteria, Elusimicrobia and Tenericutes were altered by different levels of Etn compared with the control group. An Etn concentration of 500 µm shifted colonic microbial metabolic functions that are in favour of lipid- and sugar-related metabolism and biosynthesis. Etn also altered the metabolic phenotypes such as anaerobic microbial counts, and oxidative stress tolerance at over 250 µm. This is the first report for a role of Etn in modifying gut microbiota and intestinal functions. Our findings highlighted the important role of Etn in shaping gut microbial community and promotes intestinal functions, which may provide a better insight of breast-feeding to infant's gut health.

DNA barcoding and metabarcoding of standardized samples reveal patterns of marine benthic diversity.

Documenting the diversity of marine life is challenging because many species are cryptic, small, and rare, and belong to poorly known groups. New sequencing technologies, especially when combined with standardized sampling, promise to make comprehensive biodiversity assessments and monitoring feasible on a large scale. We used this approach to characterize patterns of diversity on oyster reefs across a range of geographic scales comprising a temperate location [Virginia (VA)] and a subtropical location [Florida (FL)]. Eukaryotic organisms that colonized multilayered settlement surfaces (autonomous reef monitoring structures) over a 6-mo period were identified by cytochrome c oxidase subunit I barcoding (>2-mm mobile organisms) and metabarcoding (sessile and smaller mobile organisms). In a total area of ∼ 15.64 m(2) and volume of ∼ 0.09 m(3), 2,179 operational taxonomic units (OTUs) were recorded from 983,056 sequences. However, only 10.9% could be matched to reference barcodes in public databases, with only 8.2% matching barcodes with both genus and species names. Taxonomic coverage was broad, particularly for animals (22 phyla recorded), but 35.6% of OTUs detected via metabarcoding could not be confidently assigned to a taxonomic group. The smallest size fraction (500 to 106 µm) was the most diverse (more than two-thirds of OTUs). There was little taxonomic overlap between VA and FL, and samples separated by ∼ 2 m were significantly more similar than samples separated by ∼ 100 m. Ground-truthing with independent assessments of taxonomic composition indicated that both presence-absence information and relative abundance information are captured by metabarcoding data, suggesting considerable potential for ecological studies and environmental monitoring.