PhylOTU: a high-throughput procedure quantifies microbial community diversity and resolves novel taxa from metagenomic data

Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity?     

Analysis of human and animal fecal microbiota for microbial source tracking

Microbial compositions of human and animal feces from South Korea were analyzed and characterized. In total, 38 fecal samples (14 healthy adult humans, 6 chickens, 6 cows, 6 pigs and 6 geese) were analyzed by 454 pyrosequencing of the V2 region of the 16S rRNA gene. Four major phyla, Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, were identified in the samples. Principal coordinate analysis suggested that microbiota from the same host species generally clustered, with the exception of those from humans, which exhibited sample-specific compositions. A network-based analysis revealed that several operational taxonomic units (OTUs), such as Lactobacillus sp., Clostridium sp. and Prevotella sp., were commonly identified in all fecal sources. Other OTUs were present only in fecal samples from a single organism. For example, Yania sp. and Bifidobacterium sp. were identified specifically in chicken and human fecal samples, respectively. These specific OTUs or their respective biological markers could be useful for identifying the sources of fecal contamination in water by microbial source tracking.     

Diverse microbial communities inhabit Antarctic sponges

Genetic techniques were employed to investigate the archaeal, bacterial and eukaryotic communities associated with the Antarctic sponges Kirkpatrickia varialosa, Latrunculia apicalis, Homaxinella balfourensis, Mycale acerata and Sphaerotylus antarcticus. The phylogenetic affiliation of sponge-derived bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Denaturing gradient gel electrophoresis (DGGE) was used to determine the stability of bacterial associations within each sponge species and across spatial scales. Of the 150 archaeal clones from L. apicalis, K. varialosa and M. acerata screened by restriction fragment length polymorphism (RFLP) analysis, four unique operational taxonomic units (OTUs) were observed and all clustered closely together within the Crenarchaeota. Of the 250 sponge-derived bacterial clones screened by RFLP analysis, 61 were unique OTUs that were not detected during examination of 160 seawater-derived clones. Rarefaction analysis indicated that the clone libraries represented between 44 and 83% of the total estimated diversity. Phylogenetic analysis of sequence data revealed that the bacterial communities present in Antarctic sponges primarily clustered within the Gamma and Alpha proteobacteria and the Cytophaga/Flavobacterium of Bacteroidetes group. Bacterial DGGE analysis for replicate sponge and seawater samples at each Antarctic site revealed that bacterial communities were consistently detected within a particular species regardless of the collection site, with six bacterial bands exclusively associated with a single sponge species. Phylogenetic analysis of sequence data from eukaryotic DGGE analysis revealed that the communities present in Antarctic sponges fell into diatom and dinoflagellate clusters with many sequences having no known close relatives. In addition, seven eukaryotic sequences that were not detected in seawater samples or other sponge species were observed in K. varialosa.     

Metabarcoding of freshwater invertebrates to detect the effects of a pesticide spill

Biomonitoring underpins the environmental assessment of freshwater ecosystems and guides management and conservation. Current methodology for surveys of (macro)invertebrates uses coarse taxonomic identification where species‐level resolution is difficult to obtain. Next‐generation sequencing of entire assemblages (metabarcoding) provides a new approach for species detection, but requires further validation. We used metabarcoding of invertebrate assemblages with two fragments of the cox1 “barcode” and partial nuclear ribosomal (SSU) genes, to assess the effects of a pesticide spill in the River Kennet (southern England). Operational taxonomic unit (OTU) recovery was tested under 72 parameters (read denoising, filtering, pair merging and clustering). Similar taxonomic profiles were obtained under a broad range of parameters. The SSU marker recovered Platyhelminthes and Nematoda, missed by cox1, while Rotifera were only amplified with cox1. A reference set was created from all available barcode entries for Arthropoda in the BOLD database and clustered into OTUs. The River Kennet metabarcoding produced matches to 207 of these reference OTUs, five times the number of species recognized with morphological monitoring. The increase was due to the following: greater taxonomic resolution (e.g., splitting a single morphotaxon “Chironomidae” into 55 named OTUs); splitting of Linnaean binomials into multiple molecular OTUs; and the use of a filtration‐flotation protocol for extraction of minute specimens (meiofauna). Community analyses revealed strong differences between “impacted” vs. “control” samples, detectable with each gene marker, for each major taxonomic group, and for meio‐ and macrofaunal samples separately. Thus, highly resolved taxonomic data can be extracted at a fraction of the time and cost of traditional nonmolecular methods, opening new avenues for freshwater invertebrate biodiversity monitoring and molecular ecology.     

Considerable Bacterial Community Structure Coupling with Extracellular Electron Transfer at Karst Area Stone in Yunnan,China

Nearly all karst stones have partial dark and light color sections under the same condition. In this study, 24662 operational taxonomic units (OTUs) were examined from karst stones surface samples. Dark samples showed higher abundance of 18,115 OTUs (73.5%) and containing clustered bacterial communities, as indicated by the results of principal component and cluster analyses. Additionally, heatmap analysis showed microorganisms distinction between different color samples and 19 genera of electroactive microorganisms gathered in the dark samples. Furthermore, iron manganese oxides were detected as the main mineral composition difference. Notably, dark samples exhibited remarkable photoelectrochemical activity in response to visible light. Under sunlight, both microorganisms and minerals displayed close relationship with extracellular electron transfer process, which enhanced the understanding for microorganism-–mineral interactions in natural karst environments.     

Status of the phylogenetic diversity census of ruminal microbiomes

In this study, the collective microbial diversity in the rumen was examined by performing a meta-analysis of all the curated 16S rRNA gene (rrn) sequences deposited in the RDP database. As of November 2010, 13,478 bacterial and 3516 archaeal rrn sequences were found. The bacterial sequences were assigned to 5271 operation taxonomic units (OTUs) at species level (0.03 phylogenetic distance) representing 19 existing phyla, of which the Firmicutes (2958 OTUs), Bacteroidetes (1610 OTUs) and Proteobacteria (226 OTUs) were the most predominant. These bacterial sequences were grouped into more than 3500 OTUs at genus level (0.05 distance), but only 180 existing genera were represented. Nearly all the archaeal sequences were assigned to 943 species-level OTUs in phylum Euryarchaeota. Although clustered into 670 genus-level OTUs, only 12 existing archaeal genera were represented. Based on rarefaction analysis, the current percent coverage at species level reached 71% for bacteria and 65% for archaea. At least 78,218 bacterial and 24,480 archaeal sequences would be needed to reach 99.9% coverage. The results of this study may serve as a framework to assess the significance of individual populations to rumen functions and to guide future studies to identify the alpha and global diversity of ruminal microbiomes.     

High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples

In this methodological study, we compare 454 sequencing and a conventional cloning and Sanger sequencing approach in their ability to characterize fungal communities PCR amplified from four root systems of the ectomycorrhizal plant Bistorta vivipara. To examine variation introduced by stochastic processes during the laboratory work, we replicated all analyses using two independently obtained DNA extractions from the same root systems. The ITS1 region was used as DNA barcode and the sequences were clustered into OTUs as proxies for species using single linkage clustering (BLASTClust) and 97% sequence similarity cut-off. A relatively low overlap in fungal OTUs was observed between the 454 and the clone library datasets — even among the most abundant OTUs. In a non-metric multidimensional scaling analysis, the samples grouped more according to methodology compared to plant. Some OTUs frequently detected by 454, most notably those OTUs with taxonomic affinity to Glomales, were not detected in the Sanger dataset. Likewise, a few OTUs, including Cenococcum sp., only appeared in the clone libraries. Surprisingly, we observed a significant relationship between GC/AT content of the OTUs and their proportional abundances in the 454 versus the clone library datasets. Reassuringly, a very good consistency in OTU recovery was observed between replicate runs of both sequencing methods. This indicates that stochastic processes had little impact when applying the same sequencing technique on replicate samples.     

Identifying airborne fungi in Seoul,Korea using metagenomics

Fungal spores are widespread and common in the atmosphere. In this study, we use a metagenomic approach to study the fungal diversity in six total air samples collected from April to May 2012 in Seoul, Korea. This springtime period is important in Korea because of the peak in fungal spore concentration and Asian dust storms, although the year of this study (2012) was unique in that were no major Asian dust events. Clustering sequences for operational taxonomic unit (OTU) identification recovered 1,266 unique OTUs in the combined dataset, with between 223?96 OTUs present in individual samples. OTUs from three fungal phyla were identified. For Ascomycota, Davidiella (anamorph: Cladosporium) was the most common genus in all samples, often accounting for more than 50% of all sequences in a sample. Other common Ascomycota genera identified were Alternaria, Didymella, Khuskia, Geosmitha, Penicillium, and Aspergillus. While several Basidiomycota genera were observed, Chytridiomycota OTUs were only present in one sample. Consistency was observed within sampling days, but there was a large shift in species composition from Ascomycota dominant to Basidiomycota dominant in the middle of the sampling period. This marked change may have been caused by meteorological events. A potential set of 40 allergyinducing genera were identified, accounting for a large proportion of the diversity present (22.5?7.2%). Our study identifies high fungal diversity and potentially high levels of fungal allergens in springtime air of Korea, and provides a good baseline for future comparisons with Asian dust storms.     

Bacteria related to tick-borne pathogen assemblages in Ornithodoros cf. hasei (Acari: Argasidae) and blood of the wild mammal hosts in the Orinoquia region,Colombia

Interest in research on soft ticks has increased in recent decades, leading to valuable insight into their role as disease vectors. The use of metagenomics-based analyses have helped to elucidate ecological factors involved in pathogen, vector, and host dynamics. To understand the main bacterial assemblages present in Ornithodoros cf. hasei and its mammalian hosts, 84 ticks and 13 blood samples from bat hosts (Chiroptera) were selected, and the 16S rRNA gene V4 region was sequenced in five pools (each one related to each host-tick pairing). Bacterial taxonomic assignment analyses were performed by comparing operational taxonomic units (OTUs) shared between ticks and their host blood. This analysis showed the presence of Proteobacteria (38.8%), Enterobacteriaceae (25%), Firmicutes (12.3%), and Actinobacteria (10.9%) within blood samples, and Rickettsiaceae (39%), Firmicutes (25%), Actinobacteria (13.1%), and Proteobacteria (9%) within ticks. Species related to potentially pathogenic genera were detected in ticks, such as Borrelia sp., Bartonella tamiae, Ehrlichia sp. and Rickettsia-like endosymbiont, and the presence of these organisms was found in all analyzed bat species (Cynomops planirostris, Molossus pretiosus, Noctilio albiventris), and O. cf. hasei. About 41–48.6% of bacterial OTUs (genera and species) were shared between ticks and the blood of bat hosts. Targeted metagenomic screening techniques allowed the detection of tick-associated pathogens for O. cf. hasei and small mammals for the first time, enabling future research on many of these pathogens.

     

Developing SSU rDNA metagenomic profiles of aquatic microbial communities for environmental assessments

Five water samples from three sources, two municipal reservoirs in central North Carolina and Toolik Lake in Alaska, were processed to conduct a comparative survey of microbial small subunit rDNA sequences. Genomic DNA was extracted and amplified by PCR using universal SSU rDNA primers to generate 16S and 18S rDNA clone libraries and 50 clones from each library were sequenced and placed in operational taxonomic units (OTUs). Through this recovery and analysis of SSU rRNA genes, a metagenomic profile of the microbial community emerged for each environmental sample. Analyses of these profiles, including species diversity estimates and rank-abundance curves, revealed that approximately 64% of prokaryotic OTUs and 80% of eukaryotic OTUs were novel. Diversity estimates were consistent with predicted ecosystem characteristics: they were greater for the mesotrophic to eutrophic temperate lakes, than for the oligotrophic arctic lake. Sample comparisons showed that community similarity declined as geographic distance between sites increased. Real-time quantitative PCR results showed that OTUs which had been recovered from only one library were actually present in other samples, but at much lower frequencies, suggesting that many, if not most, microorganisms are cosmopolitan. Together, these results support the potential value of using the microbial community as an indicator of local environmental conditions. In other words, it may be realistic to monitor water quality using a single, comprehensive suite of microorganisms by analyzing patterns of relative abundance.     

Denaturing gradient gel electrophoresis analyses of the vertical distribution and diversity of Vibrio spp. populations in the Cariaco Basin

The Cariaco system is the second largest permanently anoxic marine water body in the world. Its water column is characterized by a pronounced vertical layering of microbial communities. The goal of our study was to investigate the vertical distribution and diversity of Vibrio spp. present in the Cariaco Basin waters using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments. Representatives of the Vibrio genus were detected by nested and direct PCR in seawater at 10 depths. Sequence analyses of 55 DGGE bands revealed that only 11 different operational taxonomic units (OTU) are identified as Vibrio species. Between one and five OTUs were detected at each depth and the most common OTUs were OTU 1 and OTU 2, which phylogenetically clustered with Vibrio chagasii and Vibrio fortis, respectively. OTUs 3 and 4 were only found in the anoxic zone and were identified as Vibrio orientalis and Vibrio neptunius, respectively. Several Vibrio species detected are potentially pathogenic to human, prawns and corals such as Vibrio parahaemolyticus, Vibrio fischeri and Vibrio shilonii. In the Cariaco Basin, different Vibrio species were found to be specific to specific depths strata, suggesting that this genus is a natural component of the microbial communities in this marine redox environment.     

Effects of long‐term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach

To understand the fine‐scale effects of changes in nutrient availability on eukaryotic soil microorganisms communities, a multiple barcoding approach was used to analyse soil samples from four different treatments in a long‐term fertilization experiment. We performed PCR amplification on soil DNA with primer pairs specifically targeting the 18S rRNA genes of all eukaryotes and three protist groups (Cercozoa, Chrysophyceae‐Synurophyceae and Kinetoplastida) as well as the ITS gene of fungi and the 23S plastid rRNA gene of photoautotrophic microorganisms. Amplicons were pyrosequenced, and a total of 88 706 quality filtered reads were clustered into 1232 operational taxonomic units (OTU) across the six data sets. Comparisons of the taxonomic coverage achieved based on overlapping assignment of OTUs revealed that half of the eukaryotic taxa identified were missed by the universal eukaryotic barcoding marker. There were only little differences in OTU richness observed between organic‐ (farmyard manure), mineral‐ and nonfertilized soils. However, the community compositions appeared to be strongly structured by organic fertilization in all data sets other than that generated using the universal eukaryotic 18S rRNA gene primers, whereas mineral fertilization had only a minor effect. In addition, a co‐occurrence based network analysis revealed complex potential interaction patterns between OTUs from different trophic levels, for example between fungivorous flagellates and fungi. Our results demonstrate that changes in pH, moisture and organic nutrients availability caused shifts in the composition of eukaryotic microbial communities at multiple trophic levels.     

DNA barcode‐based delineation of putative species: efficient start for taxonomic workflows

The analysis of DNA barcode sequences with varying techniques for cluster recognition provides an efficient approach for recognizing putative species (operational taxonomic units, OTUs). This approach accelerates and improves taxonomic workflows by exposing cryptic species and decreasing the risk of synonymy. This study tested the congruence of OTUs resulting from the application of three analytical methods (ABGD, BIN, GMYC) to sequence data for Australian hypertrophine moths. OTUs supported by all three approaches were viewed as robust, but 20% of the OTUs were only recognized by one or two of the methods. These OTUs were examined for three criteria to clarify their status. Monophyly and diagnostic nucleotides were both uninformative, but information on ranges was useful as sympatric sister OTUs were viewed as distinct, while allopatric OTUs were merged. This approach revealed 124 OTUs of Hypertrophinae, a more than twofold increase from the currently recognized 51 species. Because this analytical protocol is both fast and repeatable, it provides a valuable tool for establishing a basic understanding of species boundaries that can be validated with subsequent studies.     

Disparities in second‐generation DNA metabarcoding results exposed with accessible and repeatable workflows

Different second‐generation sequencing technologies may have taxon‐specific biases when DNA metabarcoding prey in predator faeces. Our major objective was to examine differences in prey recovery from bat guano across two different sequencing workflows using the same faecal DNA extracts. We compared results between the Ion Torrent PGM and the Illumina MiSeq with similar library preparations and the same analysis pipeline. We focus on repeatability and provide an R Notebook in an effort towards transparency for future methodological improvements. Full documentation of each step enhances the accessibility of our analysis pipeline. We tagged DNA from insectivorous bat faecal samples, targeted the arthropod cytochrome c oxidase I minibarcode region and sequenced the product on both second‐generation sequencing platforms. We developed an analysis pipeline with a high operational taxonomic unit (OTU) clustering threshold (i.e., ≥98.5%) followed by copy number filtering to avoid merging rare but genetically similar prey into the same OTUs. With this workflow, we detected 297 unique prey taxa, of which 74% were identified at the species level. Of these, 104 (35%) prey OTUs were detected by both platforms, 176 (59%) OTUs were detected by the Illumina MiSeq system only, and 17 (6%) OTUs were detected using the Ion Torrent system only. Costs were similar between platforms but the Illumina MiSeq recovered six times more reads and four additional insect orders than did Ion Torrent. The considerations we outline are particularly important for long‐term ecological monitoring; a more standardized approach will facilitate comparisons between studies and allow faster recognition of changes within ecological communities.     

Amplicon –Based Metagenomic Analysis of Mixed Fungal Samples Using Proton Release Amplicon Sequencing

Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs) were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn). Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific “conserved” primer pairs confirms our recommendation that a multi-region approach be taken for other amplicon-based metagenomic studies.     

Gene-targeted metagenomic analysis of glucan-branching enzyme gene profiles among human and animal fecal microbiota

Glycoside hydrolases (GHs), the enzymes that breakdown complex carbohydrates, are a highly diversified class of key enzymes associated with the gut microbiota and its metabolic functions. To learn more about the diversity of GHs and their potential role in a variety of gut microbiomes, we used a combination of 16S, metagenomic and targeted amplicon sequencing data to study one of these enzyme families in detail. Specifically, we employed a functional gene-targeted metagenomic approach to the 1-4-α-glucan-branching enzyme (gBE) gene in the gut microbiomes of four host species (human, chicken, cow and pig). The characteristics of operational taxonomic units (OTUs) and operational glucan-branching units (OGBUs) were distinctive in each of hosts. Human and pig were most similar in OTUs profiles while maintaining distinct OGBU profiles. Interestingly, the phylogenetic profiles identified from 16S and gBE gene sequences differed, suggesting the presence of different gBE genes in the same OTU across different vertebrate hosts. Our data suggest that gene-targeted metagenomic analysis is useful for an in-depth understanding of the diversity of a particular gene of interest. Specific carbohydrate metabolic genes appear to be carried by distinct OTUs in different individual hosts and among different vertebrate species'' microbiomes, the characteristics of which differ according to host genetic background and/or diet.     

Compositions and Structures of Archaeal Communities in Acid Mineral Bioleaching Systems of Dongxiang Copper Mine and Yinshan Lead–Zinc Mine, China

Four samples were studied from four separated sites with heap leaching in the Yinshan Lead-Zinc Mine and the Dongxiang Copper Mine in Jiangxi province, China. The compositions and structures of archaeal communities in four sites were identified by a polymerase chain reaction-based cloning approach. A total of six operational taxonomic units (OTUs) was obtained from four samples. The highest percentage of overlapped OTUs was 88.9% between sites DX and D1. Phylogenetic analysis revealed that archaea in the four acid mineral bioleaching systems fell into two divisions: Thermoplasma and Ferroplasma. The proportions of Thermoplasma and Ferroplasma in all four sites were 20.6% and 79.4%, respectively. The proportions of clones clustered with Ferroplasma in four sites were 93.8% (D1), 30.5% (D3), 100% (DY), and 93.2% (DX), respectively. The proportions of clones clustered with Thermoplasma in the other three sites were 6.2% (D1), 69.5% (D3), and 6.8% (DX), respectively. The results of principal component analysis based on the percentages of six OTUs obtained from four sites and geochemical data from four sites suggested that the concentrations of elements such as lead, cobalt, and sulfur might be the reason causing the different archaeal structure in site D3 than those in the other three sites.     

Clustering of fungal community internal transcribed spacer sequence data obscures taxonomic diversity

Next‐generation DNA sequencing has enabled a rapid expansion in the size of molecular fungal ecology studies employing the nuclear internal transcribed spacer (ITS) region. Many sequence‐processing pipelines and protocols require sequence clustering to generate operational taxonomic units (OTUs) based on sequence similarity as a step to reduce total data quantity and complexity prior to taxonomic assignment. However, the consequences of ITS sequence clustering in regard to sample taxonomic coverage have not been carefully examined. Here we demonstrate that typically used clustering thresholds for fungal ITS sequences result in statistically significant losses in taxonomic coverage. Analyses using environmentally derived fungal sequences indicated an average of 3.1% of species went undetected (P < 0.05) if the sequences were denoised and clustered at a 97% threshold prior to taxonomic assignment. Additionally, an in silico analysis using a reference fungal ITS database suggested that approximately 25% of species went undetected if the sequences were clustered prior to taxonomic assignment. Finally, analysis of sequences derived from pure‐cultured fungal isolates of known identity indicated sequence denoising and clustering were not critical in improving identification accuracy.     

Comparative analysis of bacterial diversity in the rhizosphere of tomato by culture-dependent and -independent approaches

The microbiome in the rhizosphere–the region surrounding plant roots–plays a key role in plant growth and health, enhancing nutrient availability and protecting plants from biotic and abiotic stresses. To assess bacterial diversity in the tomato rhizosphere, we performed two contrasting approaches: culture-dependent and -independent. In the culture-dependent approach, two culture media (Reasoner’s 2A agar and soil extract agar) were supplemented with 12 antibiotics for isolating diverse bacteria from the tomato rhizosphere by inhibiting predominant bacteria. A total of 689 bacterial isolates were clustered into 164 operational taxonomic units (OTUs) at 97% sequence similarity, and these were found to belong to five bacterial phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Firmicutes). Of these, 122 OTUs were retrieved from the antibiotic-containing media, and 80 OTUs were recovered by one specific antibiotic-containing medium. In the culture-independent approach, we conducted Illumina MiSeq amplicon sequencing of the 16S rRNA gene and obtained 19,215 high-quality sequences, which clustered into 478 OTUs belonging to 16 phyla. Among the total OTUs from the MiSeq dataset, 22% were recovered in the culture collection, whereas 41% of OTUs in the culture collection were not captured by MiSeq sequencing. These results showed that antibiotics were effective in isolating various taxa that were not readily isolated on antibiotic-free media, and that both contrasting approaches provided complementary information to characterize bacterial diversity in the tomato rhizosphere.     

Fungal communities in wheat grain show significant co-existence patterns among species

The wheat grain mycobiome is only scarcely investigated and focus has been on seed-transmitted wheat pathogens of agricultural importance. In this study, we used next generation sequencing to study the mycobiome of Danish wheat grain samples at harvest. In total 228,421 sequences were obtained from 90 samples that were taken from locations across Denmark during three years. These sequences could be grouped into 173 non-singleton operational taxonomic units (OTUs) of which 21 OTUs, identified as belonging to genera such as Fusarium, Alternaria, Cladosporium. Phaeosphaeria and Microdochium, were identified as ‘core’ OTUs as they were found in all or almost all samples and accounted for almost 99 % of all sequences. The remaining OTUs were only sporadically found and only in small amounts. Cluster and factor analyses showed patterns of co-existence among the core species. Cluster analysis grouped the 21 core OTUs into three clusters: cluster 1 consisting of saprotrophs, cluster 2 consisting mainly of yeasts and saprotrophs and cluster 3 consisting of wheat pathogens. Principal component extraction showed that the Fusarium graminearum group was inversely related to OTUs of clusters 1 and 2.