High-throughput Sequencing Approach Research Articles

Spatial and temporal distribution of aerobic anoxygenic phototrophic bacteria: key functional groups in biological soil crusts.

Several significant ecosystem services are performed by biological soil crusts (BSCs) in drylands, wherein photoautotrophic microorganisms are commonly critical contributors. However, aerobic anoxygenic phototrophic bacteria (AAnPB) are rarely reported in BSCs, despite being the second major branch of Earth's phototrophic microbes. Here, we collected different types of BSCs and their subsoils from temperate deserts, investigated distributions of AAnPB communities among BSCs using cultivation and high-throughput sequencing approaches, predicted keystone species by co-occurrence network analysis, and verified their effects on BSCs formation through microcosm experiments. The absolute abundances and diversity of AAnPB were higher in BSCs and were closely related with BSCs successional stages, as well as soil organic carbon contents. AAnPB communities in both BSCs and their subsoils were dominated by Proteobacteria and Alphaproteobacteria, specifically Acetobacteraceae, Rhodospirillaceae, Roseiflexaceae, Sphingomonadaceae and Caulobacteraceae families. Mean annual precipitation, pH and available nutrients were the primary factors that shaped AAnPB community structures. The predicted keystone species belonged to the families Acetobacteraceae, Rhodospirillaceae and Sphingomonadanceae. Microcosm experiments demonstrated that inoculation with strains from the three families greatly accelerated the formation and development of BSCs. These observations suggest that AAnPB are likely important functional groups in BSCs that significantly contribute to their formation and important ecosystem services.

MicroRNA miR-8 promotes cell growth of corpus allatum and juvenile hormone biosynthesis independent of insulin/IGF signaling in Drosophila melanogaster

The Drosophila melanogaster corpus allatum (CA) produces and releases three types of sesquiterpenoid hormones, including juvenile hormone III bisepoxide (JHB3), juvenile hormone III (JH III), and methyl farnesoate (MF). JH biosynthesis involves multiple discrete enzymatic reactions and is subjected to a comprehensive regulatory network including microRNAs (miRNAs). Using a high throughput sequencing approach, we have identified abundant miRNAs in the D. melanogaster ring gland, which consists of the CA, prothoracic gland, and corpus cardiaca. Genetic and qPCR screens were then performed in an attempt to uncover the full repertoire of CA miRNAs that are involved in regulating metamorphosis. miR-8 was identified as a potential candidate and further studied for its role in the CA. Overexpression of miR-8 in the CA increased cell size of the gland and expression of Jhamt (a gene coding for a key regulatory enzyme in JH biosynthesis), resulting in pupal lethality. By contrast, sponge-mediated reduction of miR-8 in the CA decreased cell size and Jhamt expression, but did not cause lethality. Further investigation revealed that miR-8 promotes cell growth independent of insulin/IGF signaling. Taken together, these experiments show that miR-8 is highly expressed in the CA and exerts its positive effects on cell growth and JH biosynthesis. The miRNAs data in the ring gland also provide a useful resource to study how miRNAs collaboratively regulate hormone synthesis in D. melanogaster.

High Throughput Sequencing in 3449 Patients with Bleeding and Platelet Disorders: Novel Gene Discovery and Robust Diagnosis

BackgroundBleeding symptoms are common and reported at some point by 25% of the population. Laboratory investigations frequently fail to provide an explanation for the symptoms or to identify those at significant risk of future bleeding. A similar problem is presented by patients with congenital thrombocytopenia which often cannot be distinguished from acquired disorders and making investigation of platelet function impractical. The broad heterogeneity of phenotypes associated with established disorders further complicates diagnosis. For these reasons, we have developed High Throughput Sequencing (HTS) approaches to investigate patients with undiagnosed bleeding and platelet disorders (BPDs) through the ThromboGenomics 79 gene panel HTS (TG) test and by whole genome sequencing (WGS).MethodsBPD cases were recruited in 3 broad categories: Group 1: 1321 cases suspected to have a defect in one of the 79 known BPD genes (ie genes known to harbour variants responsible for a BPD); Group 2: 212 cases being prepared for elective surgery with self-reported bleeding symptoms; and Group 3: 1916 cases with a suspected inherited BPD, not thought to be caused by a variant in one of the 79 known BPD genes. DNA samples from the first two groups were sequenced by the TG test and from the third group by WGS as part of the first 35,000 samples for the 100,000 Genomes Project.In total, 3449 DNA samples from BPD patients have been analysed by HTS. Laboratory and clinical phenotypes were recorded using Human Phenotype Ontology (HPO) terms. Variants were called and prioritised based on minor allele frequency, predicted impact and presence in the Human Gene Mutation Database and those variants present in the 79 known BPD genes were reviewed by a multi-disciplinary team (MDT) where pathogenicity was assigned to variants using ACGS criteria (Clearly pathogenic [CPV], Likely pathogenic [LPV] and Variant of unknown significance [VUS]).ResultsA mean of 6.6 variants were assessed at MDT for each case. In Group 1 pathogenic variants were observed in 713 of 1321 (54%) of cases and >40% were novel and labelled as LPV; ~20% of variants discussed were designated VUS. In sharp contrast, pathogenic variants were only identified in 6 of 212 (2.8%) Group 2 cases. In Group 3, pathogenic variants were identified in 106 of 1916 (12%) cases. In >300 cases with non-syndromic thrombocytopenia, CPV or LPV were identified in ~30%. In contrast, in 131 cases with a significant bleeding disorder but no identified laboratory platelet or coagulation abnormality causal variants were observed in only 5 cases.ConclusionThese results demonstrate the success of HTS approaches in providing a genetic diagnosis for patients with well-defined inherited platelet or coagulation defects. In addition, 23 novel BPD genes were identified by analysis of the WGS data from Group 3 cases, including NBEAL2, RBM8A, SRC, DIAPH1, TPM4, ABCC4 and KDSR. Furthermore extensive replication was demonstrated in nearly 100 Group 3 cases for recent BPD gene discoveries by other groups (C6ORF25, CYCS, RNU4ATAC, STIM1, RASGRP2, ETV6, ACTN1). All MDT-reviewed variants and appended HPO terms are shared in ClinVar, supporting the international effort to improve reference catalogues, aligned to ASH's Precision Medicine initiative.DiscussionHTS has been particularly successful for thrombocytopenia, but the yield for bleeding disorders without numerical platelet or coagulation defects is low. Our results, and the observations by others are compatible with the notion that the genetic architecture of unresolved BPD cases is extremely diverse. Continuing efforts such as the 100,000 Genomes Project with sharing and transparency of data across projects will enable better interpretation of variants and the discovery of novel genes. Finally, we are annotating the non-coding space with data from recently identified GWAS variants for blood cell traits, BLUEPRINT and ROADMAP epigenome marks, and eQTLs for blood cell traits to interpret the regulatory regions of the genome and identify non-coding variants causing BPD and other unresolved rare diseases of the blood. DisclosuresNo relevant conflicts of interest to declare.

Transcriptome of DNA Repair Genes in CMML: Rationale for Targeting ERCC1 and CDKN1A As a Synthetic Lethality Approach

Background: Chronic myelomonocytic leukemia (CMML) is characterized by features of both myeloproliferative neoplasm and myelodysplastic syndrome (MDS). Despite the main role of DNA repair pathways in cancer, in myelodysplastic disorders, somatic mutations are not found in these pathways. The novel discovery of non-classical leukaemogenesis mediated by splicing defects, profound influence of epigenetic anomalies, deeper knowledge of the DNA repair network, and development of unbiased high-throughput sequencing approaches, prompted us to revisit whether these processes might be disrupted by non-mutational mechanisms in CMML and related myeloid disorders. Study aims: i) to screen the transcriptome of DNA repair genes in a CMML discovery cohort; ii) to validate their misregulation in an independent large CMML cohort; and iii) determining how those candidates behave through the spectrum of myeloid malignancies. Methods: RNA-seq was performed in the discovery cohort: 27 CMML bone marrow samples (BM) at diagnosis and 9 healthy BM samples, and in 7 patients treated with Azacitidine in BM collected before and after 4 cycles. RT-qPCR was used to validate 9 DEGs through the myeloid spectrum and in a CMML validation cohort: 74 additional CMML, 70 MDS, 66 AML , and 25 CML patients. Candidates were chosen based on clinical considerations: i) druggable oncogenes highly overexpressed ii) oncogenes infra-expressed with inhibitory molecules already being tested in myeloid neoplasms. Global pattern of DNA repair gene expression was compared with MDS and AML extracted from MILE study data. The DEGs methylation status was studied using available public data and we confirmed by pyrosequencing the methylation status of our main candidates. The CMML validation cohort was mutationally characterized by targeted sequencing of 18 CMML recurrently mutated genes. Alternative splice sites, skipped exons, mutually exclusive exons and retained introns were quantified using rMATS.Results: Of 27 CMML patients and 9 healthy donors, the expression of 30 genes was significantly different between the two groups (8 genes up-regulated and 22 down-regulated). Defects on genes predominantly unique to a single strand breaks repair pathway included: NEIL1 and OGG1 in base excision repair, XPA and MSS19 in nucleotide excision repair and RPA4 in mismatch repair. XRCC4 and MSH4 overexpression and PRKDC down-regulation were found as significant changes among the genes associated exclusively to double strand breaks repair. We next extracted the DNA repair transcriptional components from MILE study: 206 MDS, 47 ckAML and 73 healthy donors. MDS misregulation was characterized by upregulated genes while ckAML showed a global disruption, mostly downregulation of Homologous Recombination genes. Some genes showed opposite sense of misregulation depending on the myeloid entity: TDP1 was upregulated in CMML cases and downregulated in ckAML, and viceversa for BAP1 . CDK1 and EXO1 were upregulated in MDS cases but downregulated in ckAML cases. Statistically significant and direction-concordant dysregulation of CDKN1A, ERCC1, XRCC4, NEIL1, MSH4, PARP1 and BAP1 were confirmed in our independent CMML validation cohort. The methylation study showed a global demethylated status of our misregulated DNA repair genes in CMML. Either responder s (n=3) or non-responders (n=2) to azacitidine failed to achieve a substantial expression change in their DNA Repair DEGs. PARP1 infraexpresion was associated with TET2 mut cases. Lower CDKN1A and BAP1 expression was related to chromatin regulator mutations. SRSF2 mutations induced widespread aberrant splicing events in DNA repair genes in CMMLConclusions: We have identified by means of an unbiased high-throughput approach, and validated in an independent cohort, a subset of DNA repair genes consistently misregulated in CMML. The genes identified warrant further study as potential novel therapeutic targets, both directly and through modulation of associated compensatory pathways. CDKN1A and ERCC1 in particular emerge as realistic candidates for a synthetic lethality approach. Our findings suggest that the promise of PARP1 inhibition appears less pertinent to CMML than for other myeloid diseases. These differences might also partially explain the different genomic and phenotypic manifestations of different myeloid neoplasms, and shed insights into their distinctive biology. DisclosuresMaciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy.

Rapid Capture NGS Identifies Genomic Rearrangements in BCP-ALL for MRD Diagnostics

BackgroundImmunoglobin heavy chain (IGH), T-cell receptor δ (TRδ) rearrangements and translocations have frequently been described in patients with B-cell precursor (BCP) ALL. In order to detect the various types of fusions we used a high-throughput sequencing (HTS) assay based on direct capturing and sequencing of enriched genomic DNA fragments. Our approach focused on enrichment and direct paired-end sequencing of IG/TR rearrangements to analyze clonality and to detect kinase- and/or cytokine-receptor fusion breakpoints. We selected the corresponding chromosomal regions spanning exonic as well as intronic genomic areas from IGH-JH, TRδ and from several “Ph-like”-associated genes (ABL1, ABL2, CSF1R, CRLF2, EPOR, JAK2, PDGFRß). For the identification of clonality and fusion breakpoints we used Vidjil (www.Vidjil.org) and “segemehl” bioinformatics algorithms, respectively. Clonal rearrangements and genomic breakpoints were subsequently compared for their use in detection of minimal residual disease (MRD).Patients and methodsFor combined clonality and breakpoint analysis, 162 BCP ALL patients (MLL-; TEL-AML1-, BCR-ABL1-) were selected due to the MRD status at the end of induction (EOI) therapy, which include a substantial proportion of ALL samples (n = 68) with a high MRD burden (≥ 1 x 10-2). To test the feasibility of the analytical approach, additional BCR-ABL1+ ALLsamples (n=19) were included for genomic breakpoint analysis. Captured genomic DNA from eighty ALL samples was sequenced on a HiSeq2500 Illumina device, the remaining samples were subjected to several MiSeq (Illumina) runs multiplexing up to 12 samples using MiSeq Reagent Kit v3 (600 cycle) chemistry.ResultsFor TRδ, a total of 267 clonal rearrangements were characterized in 142/162 samples (88%). Beside clonal Vδ2-Dδ3, Dδ2-Dδ3, Vδ2-Jα, we identified a novel group of recurrent Dδ-Jα rearrangements in 46 samples (28%), comprising Dδ2 or Dδ3 segments fused predominantly to Jα29. For IGH-JH, 304 clonal rearrangements were identified in 152/162 samples, including several novel VDJ joinings usually not detectable by standard PCR-based approaches. In BCR-ABL1+ ALL, genomic breakpoints were identified in 17/19 cases. In BCP-ALL lacking a classical rearrangement, 50 gene fusions were detected, between IGH-JH and either DUX4, EPOR or CRLF2 in 21 of 162 cases. In addition, EBF1-PDGFRß or PAR1 deletions were detected in 5 and 10 cases, respectively. Prolonged MRD was observed in ALL carrying CRLF2rearr, EPORrearr -, PDGFR-ßrearr and as an unanticipated finding in 8/10 DUX4rearr patients who mainly underwent hematopoietic stem cell transplantation in first remission (n=5) or had a relapse (n=2). MLPA revealed a non-deleted ERG status in these 8 DUX4rearr ALL samples. A distinct subgroup of MRD high-risk patients were characterized by a complete lack of IGH-VDJ rearrangements, but they exhibited frequent IKZF1del (7/10) and a variety of other genomic rearrangements (n=3 EPORrearr, n=3 DUX4rearr, n=1 CRLF2rearr, n=1 EBF1-PDGFRß or n=1 FOXP1-ABL1). Among gene-fusion positive ALLs, breakpoint- specific real-time quantitative MRD assays at EOI were established, which demonstrated equivalent or superior sensitivity and specificity compared to conventional TR/IG targeted MRD measurements.ConclusionsThe HTS approach presented here yields classical IGH/TRδ-related clonal MRD targets and genomic breakpoints from gene fusions at a time, the latter of which can subsequently serve as robust markers for follow-up monitoring. The MRD high-risk group (≥ 1 x 10-2 at EOI) revealed an enrichment of aberrant gene fusions/rearrangements in 31 of 68 (46%) patients compared to 19 of 94 (20%) in the moderate-risk MRD group (>1 x 10-4 < 1 x 10-2). Rapid capture sequencing in a clinical diagnostics setting allows for specific ABL or JAK-STAT pathway directed early intervention in the majority of gene-fusion positive patients. DisclosuresNo relevant conflicts of interest to declare.

Comparing Quantitative Methods for Analyzing Sediment DNA Records of Cyanobacteria in Experimental and Reference Lakes.

Sediment DNA (sedDNA) analyses are rapidly emerging as powerful tools for the reconstruction of environmental and evolutionary change. While there are an increasing number of studies using molecular genetic approaches to track changes over time, few studies have compared the coherence between quantitative polymerase chain reaction (PCR) methods and metabarcoding techniques. Primer specificity, bioinformatic analyses, and PCR inhibitors in sediments could affect the quantitative data obtained from these approaches. We compared the performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the quantification of target genes of cyanobacteria in lake sediments and tested whether the two techniques similarly reveal expected patterns through time. Absolute concentrations of cyanobacterial 16S rRNA genes were compared between ddPCR and HTS using dated sediment cores collected from two experimental (Lake 227, fertilized since 1969 and Lake 223, acidified from 1976 to 1983) and two reference lakes (Lakes 224 and 442) in the Experimental Lakes Area (ELA), Canada. Relative abundances of Microcystis 16S rRNA (MICR) genes were also compared between the two methods. Moderate to strong positive correlations were found between the molecular approaches among all four cores but results from ddPCR were more consistent with the known history of lake manipulations. A 100-fold increase in ddPCR estimates of cyanobacterial gene abundance beginning in ~1968 occurred in Lake 227, in keeping with experimental addition of nutrients and increase in planktonic cyanobacteria. In contrast, no significant rise in cyanobacterial abundance associated with lake fertilization was observed with HTS. Relative abundances of Microcystis between the two techniques showed moderate to strong levels of coherence in top intervals of the sediment cores. Both ddPCR and HTS approaches are suitable for sedDNA analysis, but studies aiming to quantify absolute abundances from complex environments should consider using ddPCR due to its high tolerance to PCR inhibitors.

Forest plant cover and mineral type determine the diversity and composition of mineral-colonizing fungal communities

Soil fungi play a fundamental role in the cycling of nutrients and tree nutrition in forests. As mycorrhizal fungi are closely associated with their host trees and provide them essential base cations and phosphorus, we hypothesize that they actively mine soil minerals and transfer their nutritive content to their host. Therefore, mycorrhizal fungi are expected to be important colonizers of soil mineral surfaces relative to other fungal trophic guilds. To test this hypothesis, 50 μm nylon mesh bags were filled with three different mineral types (i.e. calcite, apatite and obsidian) and buried in the same soil colonized by three forest stands (i.e. beech, Corsican pine and coppice with standards). After an in situ incubation period of 29 months in the topsoil, mineral dissolution was detectable in the mesh bags, and the analysis of the fungal communities was therefore undertaken. Minerals presented a lower fungal biomass than the surrounding bulk soil and no difference between the three mineral types tested was observed. The high-throughput sequencing (HTS) approach showed that the fungal communities differed among the three forest stands, but in each case, mineral associated fungal taxa differed significantly from those in the bulk soil. To a lesser extent, fungal communities further varied between the three mineral types. Overall, trophic inference analyses suggested that two groups of plant-associated fungi, namely the ectomycorrhizal and endophytic fungi, were the major fungal mineral colonizers. Saprophytes also appeared significantly enriched on minerals comparatively to the surrounding bulk soil. Together, our data reveal at local scale that the mineral-associated fungal communities are first determined by the forest plant cover and then by the type of mineral.

In-depth assessment of the PAM compatibility and editing activities of Cas9 variants.

A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR–Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR–Cas9 editing, raising an urgent issue to be addressed.

Diversity and activities of pioneer bacteria, algae, and fungi colonizing ceramic roof tiles during the first year of outdoor exposure

Like other building materials, ceramic roof tiles are biodeteriorated over time. Natural conditions affect deterioration processes, but the development of a biofilm composed of many types of microorganisms also plays a critical role in biodeterioration. However, the pioneering stages of biofilm formation remain poorly studied, whereas they determine the development of the well-characterized mature biofilms. To further characterize this pioneering microbial diversity, the bacterial, algal, and fungal colonization of new tiles exposed to outdoor conditions over one full year was monitored, combining fluorimetry, biofilm imaging (SEM, confocal macroscopy), culture-based, and high-throughput sequencing approaches. For all the samples, pioneering biofilms were found to be extremely diverse but dominated by Actinobacteria (Pantoea sp. - up to 90% of the bacterial diversity -, and Pseudomonas sp. -up to 75%-), Chlorophyta (Trebouxiophyceae -100% of the algal diversity-), and Pleosporales (Alternaria sp. -up to 75% of the fungal diversity-). Interestingly, if biofilm development was highly heterogeneous between tiles and exposition sites, microbial colonization patterns succession remained comparable regardless of tile type and exposure site. Additionally, we investigated whether microbial isolates collected in this study harbor some physiological traits related to their pioneer character. This work revealed that pioneer organisms colonizing tiles harbored interesting features like the ability to produce biofilm matrix and anti-quorum sensing and antimicrobial compounds, highlighting the intense competition between the microbial players during the priming of the tile colonization. Altogether, our results emphasize the necessity to taxonomically and functionally characterize the key pioneering microorganisms involved in biofilm formation to design innovative antifouling solutions for ceramic roof tiles.

The complete chloroplast genome sequence of Strobilanthes tonkinensis Lindau

Strobilanthes tonkinensis Lindau is a member of the family Acanthaceae, which was originated from Yunnan province of China and is used as tea and health promotion. Here, we reported the complete chloroplast genome sequence of S. tonkinensis using Illumina high-throughput sequencing approach. The size of the chloroplast genome is 144,765 bp in length, containing a pair of inverted repeats (IRs, 17,362 bp) that are separated by the large single-copy (LSC, 92,248 bp), and small single-copy (SSC, 17,793 bp) regions. A total of 129 genes were identified, including 37 tRNA genes, 8 rRNA genes, and 84 protein-coding genes. The overall GC content is 38.21%. Phylogenetic analysis indicated that S. tonkinensis is closely related to Strobilanthes cusia and Strobilanthes bantonensis.

Repeat proliferation and partial endoreplication jointly shape the patterns of genome size evolution in orchids.

Although the evolutionary drivers of genome size change are known, the general patterns and mechanisms of plant genome size evolution are yet to be established. Here we aim to assess the relative importance of proliferation of repetitive DNA, chromosomal variation (including polyploidy), and the type of endoreplication for genome size evolution of the Pleurothallidinae, the most species-rich orchid lineage. Phylogenetic relationships between 341 Pleurothallidinae representatives were refined using a target enrichment hybrid capture combined with high-throughput sequencing approach. Genome size and the type of endoreplication were assessed using flow cytometry supplemented with karyological analysis and low-coverage Illumina sequencing for repeatome analysis on a subset of samples. Data were analyzed using phylogeny-based models. Genome size diversity (0.2-5.1 Gbp) was mostly independent of profound chromosome count variation (2n=12-90) but tightly linked with the overall content of repetitive DNA elements. Species with partial endoreplication (PE) had significantly greater genome sizes, and genomic repeat content was tightly correlated with the size of the non-endoreplicated part of the genome. In PE species, repetitive DNA is preferentially accumulated inthe non-endoreplicated parts of their genomes. Our results demonstrate that proliferation of repetitive DNA elements and PE together shape thepatterns of genome size diversity in orchids.

Warming and drought increase but wetness reduces the net sink of CH4 in alpine meadow on the Tibetan Plateau

Alpine meadows are significant CH4 sink. However, the response of soil CH4 uptake to future climate change in the alpine meadows remains poorly understood. We carried out an alpine meadow field experiments to determine how warming and altered precipitation (drought and wetness) affect CH4 uptake, as well as its underlying mechanisms. Our results showed that this mesic alpine meadow was a CH4 sink under all treatments (ranging from −311.4 to −487.1 mg m−2 CH4). Warming significantly increased CH4 uptake by an average of 18.14% of the annual cumulative CH4 uptake and by 48.68% of the non-growing season cumulative CH4 uptake. Drought enhanced the positive warming effect on CH4 uptake by decreasing soil water content, but wetness weakened this effect. Moreover, using high-throughput sequencing and GeoChip approaches, we found that warming stimulated CH4 uptake not only by increasing CH4 oxidation but also by decreasing CH4 production through changing the methanotroph community compositional structure. Further analysis revealed that the dynamics of daily CH4 uptake were mainly regulated by soil water content during the growing season, whereas daily CH4 uptake was strongly correlated with soil temperature during the non-growing season. Warming and altered precipitation influence soil CH4 uptake in different ways during the growing and non-growing seasons. Based on our results, we predict that Tibetan Plateau mesic alpine meadows will remain a net sink of CH4 under future climate warming and drought scenarios, but soil CH4 uptake will decrease if precipitation increases in future.

Variable response of arsenic contaminated groundwater microbial community to electron acceptor regime revealed by microcosm based high-throughput sequencing approach

Arsenic (As) mobilization in alluvial aquifers is facilitated by microbially catalyzed redox transformations that depend on the availability of electron acceptors (EAs). In this study, the response of an As-contaminated groundwater microbial community from West Bengal, India towards varied EAs was elucidated through microcosm based 16S rRNA gene amplicon sequencing. Acinetobacter, Deinococcus, Nocardioides, etc., and several unclassified bacteria (Ignavibacteria) and archaea (Bathyarchaeia, Micrarchaeia) previously not reported from As-contaminated groundwater of West Bengal, characterized the groundwater community. Distinct shifts in community composition were observed in response to various EAs. Enrichment of operational taxonomic units (OTUs) affiliated to Denitratisoma (NO3ˉ), Spirochaetaceae (Mn4+), Deinococcus (As5+), Ruminiclostridium (Fe3+), Macellibacteroides (SO4 2ˉ), Holophagae-Subgroup 7 (HCO3ˉ), Dechloromonas and Geobacter (EA mixture) was noted. Alternatively, As3+ amendment as electron donor allowed predominance of Rhizobium. Taxonomy based functional profiling highlighted the role of chemoorganoheterotrophs capable of concurrent reduction of NO3ˉ, Fe3+, SO4 2ˉ, and As biotransformation in As-contaminated groundwater of West Bengal. Our analysis revealed two major aspects of the community, (a) taxa selective toward responding to the EAs, and (b) multifaceted nature of taxa appearing in abundance in response to multiple substrates. Thus, the results emphasized the potential of microbial community members to influence the biogeochemical cycling of As and other dominant anions/cations.

Denitrifier abundance and community composition linked to denitrification potential in river sediments.

Denitrification in river sediments plays a very important role in removing nitrogen in aquatic ecosystem. To gain insight into the key factors driving denitrification at large spatial scales, a total of 135 sediment samples were collected from Huaihe River and its branches located in the northern of Anhui province. Bacterial community composition and denitrifying functional genes (nirS, nirK, and nosZ) were measured by high-throughput sequencing and real-time PCR approaches. Potential denitrification rate (PDR) was measured by acetylene inhibition method, which varied from 0.01 to 15.69 μg N g-1 h-1. The sequencing results based on 16S rRNA gene found that the main denitrification bacterial taxa included Bacillus, Thiobacillus, Acinetobacter, Halomonas, Denitratisoma, Pseudomonas, Rhodanobacter, and Thauera. Therein, Thiobacillus might play key roles in the denitrification. Total nitrogen and N:P ratio were the only chemical factors related with all denitrification genes. Furthermore, nirS gene abundance could be more susceptible to environmental parameters compared with nirK and nosZ genes. Canonical correspondence analysis indicated that NO3-, NO2-, NH4+ and IP had the significant impacts on the nirS-encoding bacterial community and spatial distributions. There was a significantly positive correlation between Thiobacillus and nirS gene. We considered that higher numbers of nosZ appeared in nutrient rich sediments. More strikingly, PDR was positively correlated with the abundance of three functional genes. Random forest analysis showed that NH4+ was the most powerful predictor of PDR. These findings can yield practical and important reference for the bioremediation or evaluation of wetland systems.

Tapirus bairdii-Associated Fecal Microbiome from a Critical Conservation Area: Calakmul, México.

Baird's tapir (Tapirus bairdii) is the largest native terrestrial mammal in the Neotropics, which is endangered primarily as a consequence of habitat loss and overhunting. Baird's tapir is predominantly nocturnal and exists at low densities which complicates field studies. Baird's tapir is a large-bodied herbivore that plays a key role in maintaining healthy tropical forests through seed dispersal in its feces. Studies of gut microbiome are essential and valuable to assess the health status of the host and the interaction with the environment. In this study, we collected fresh fecal samples of T. bairdii to analyze its gut microbiome during the rainy and dry seasons in the Calakmul region, which is a critical rainforest conservation area in Mexico. The results of a high-throughput 16S rDNA gene sequencing approach suggest that the fecal microbiome of Baird's tapir has no significant differences in composition among seasons. The most common phyla were Firmicutes, Bacteroidetes, Proteobacteria, Kiritimatiellaeota, and Spirochaetes. This study suggests that the stability of the fecal microbiome is related to similar feeding strategies throughout the year, and emphasizes the value of tapir in seed dispersal (and associated microbes) to the well-conserved forests of the Greater Calakmul region as biodiversity hotspots for conservation.

Microbial Composition of SCOBY Starter Cultures Used by Commercial Kombucha Brewers in North America.

Kombucha fermentation is initiated by transferring a solid-phase cellulosic pellicle into sweetened tea and allowing the microbes that it contains to initiate the fermentation. This pellicle, commonly referred to as a symbiotic culture of bacteria and yeast (SCOBY), floats to the surface of the fermenting tea and represents an interphase environment, where embedded microbes gain access to oxygen as well as nutrients in the tea. To date, various yeast and bacteria have been reported to exist within the SCOBY, with little consensus as to which species are essential and which are incidental to Kombucha production. In this study, we used high-throughput sequencing approaches to evaluate spatial homogeneity within a single commercial SCOBY and taxonomic diversity across a large number (n = 103) of SCOBY used by Kombucha brewers, predominantly in North America. Our results show that the most prevalent and abundant SCOBY taxa were the yeast genus Brettanomyces and the bacterial genus Komagataeibacter, through careful sampling of upper and lower SCOBY layers. This sampling procedure is critical to avoid over-representation of lactic acid bacteria. K-means clustering was used on metabarcoding data of all 103 SCOBY, delineating four SCOBY archetypes based upon differences in their microbial community structures. Fungal genera Zygosaccharomyces, Lachancea and Starmerella were identified as the major compensatory taxa for SCOBY with lower relative abundance of Brettanomyces. Interestingly, while Lactobacillacae was the major compensatory taxa where Komagataeibacter abundance was lower, phylogenic heat-tree analysis infers a possible antagonistic relationship between Starmerella and the acetic acid bacterium. Our results provide the basis for further investigation of how SCOBY archetype affects Kombucha fermentation, and fundamental studies of microbial community assembly in an interphase environment.

Virus Surveys in Olive Orchards in Greece Identify Olive Virus T, a Novel Member of the Genus Tepovirus.

Field surveys were conducted in Greek olive orchards from 2017 to 2020 to collect information on the sanitary status of the trees. Using a high-throughput sequencing approach, viral sequences were identified in total RNA extracts from several trees and assembled to reconstruct the complete genomes of two isolates of a new viral species of the genus Tepovirus (Betaflexiviridae), for which the name olive virus T (OlVT) is proposed. A reverse transcription–polymerase chain reaction assay was developed which detected OlVT in samples collected in olive growing regions in Central and Northern Greece, showing a virus prevalence of 4.4% in the olive trees screened. Sequences of amplified fragments from the movement–coat protein region of OlVT isolates varied from 75.64% to 99.35%. Three olive varieties (Koroneiki, Arbequina and Frantoio) were infected with OlVT via grafting to confirm a graft-transmissible agent, but virus infections remained latent. In addition, cucumber mosaic virus, olive leaf yellowing-associated virus and cherry leaf roll virus were identified.

Aquatic microbial community is partially functionally redundant: Insights from an in situ reciprocal transplant experiment

Microbial communities are considered to be functionally redundant, but few studies have tested this hypothesis empirically. In this study, we performed an in situ reciprocal transplant experiment on the surface and bottom waters of two lakes (Tsuei-Feng (T) and Yuan-Yang (Y)) with disparate trophic states and tracked changes in their microbial community composition and functions for 6 weeks using high-throughput sequencing and functional approaches. T lake's surface (Ts) and bottom (Tb) water active bacterial community (16S rRNA gene-transcript) was dominated by Actinobacteria, Bacteroidia, and Cyanobacteria, whereas Y lake's surface (Ys) and bottom (Yb) water had Gammaproteobacteria, Alphaproteobacteria, and Bacteroidia as the dominant classes. The community composition was resistant to changes in environmental conditions following the reciprocal transplant, but their functions tended to become similar to the incubating lakes' functional profiles. A significant linear positive relationship was observed between the microbial community and functional attributes (surface: R2 = 0.5065, p < 0.0001; bottom: R2 = 0.4592, p < 0.0001), though with varying scales of similarity (1-Bray Curtis distance), suggesting partial functional redundancy. Also, the entropy-based L-divergence measure identified high divergence in community composition (surface: 1.21 ± 0.54; bottom: 1.17 ± 0.51), and relatively low divergence in functional attributes (surface: 0.04 ± 0.01; bottom: 0.04 ± 0.01) in the two lakes' surface and bottom waters, providing further support for the presence of partial functional redundancy. This study enriches our understanding of community functional relationships and establishes the presence of partial functional redundancy in freshwater ecosystems.

Targeted amplicon sequencing reveals the probiotic potentials of microbial communities associated with traditional fermented foods of northeast India

Ethnic fermented foods are being consumed from the ancient times with a belief of having health benefits and could serve as potential source of probiotics; but there is little understanding of the microorganisms performing the fermentation processes. Apart from these benefits occasional acquisition of pathogenic microorganisms may pose health risks. Further, most of the marketed probiotic strains generally are non-indigenous in nature, thus the efficacy in a population is questionable. Hence, the present study attempts to decipher the microbial communities associated with the ethnic fermented food from northeast part of India, using high throughput sequencing approach. We noted food specific enrichment of bacterial genera viz. Lactobacillus (90.7%) in ‘Axone’, Bacillus (62%) and Clostridium (15.9%) in ‘Bastenga’ and, Lactobacillus (47.8%) and Staphylococcus (36.3%) in ‘Chathur’, whereas Saccharomyces (98.7%) belonging to phylum Ascomycota predominates the fungal communities among all the samples. Assessment of probiotic potential inferred using PICRUSt2 suggests differential abundance of genes featuring active removal of stressors, cell envelope and cell surface proteins enabling gut survival by microbial communities. Interestingly, fungal communities showed difference prevalence of luxS gene catering anti-pathogenic effect. This, represents an opportunity to identify an indigenous probiotic candidate.

Long-term effects of maize straw return and manure on the microbial community in cinnamon soil in Northern China using 16S rRNA sequencing.

Excessive use of chemical fertilizers in agricultural practices have demonstrated a significant impact on microbial diversity and community in soil by altering soil physical and chemical properties, thereby leading to a certain degree of soil salinization and nutritional imbalances. As an organic amendment, maize straw has been widely used to improve soil quality; however, its effect on the soil bacterial community remains limited in Calcarie-Fluvie Cambisols soil in semi-humid arid plateau of North China. In the present experiment, we investigated the effects of continuous straw utilization and fertilization on bacterial communities in Shouyang, Shanxi province, China. Soil samples were collected from 5 different straw utilization and fertilization modes in the following ways: straw mulching (SM), straw crushing (SC), cattle manure (CM), in which way straw is firstly used as silage and then organic fertilizer, control with no straw return (NSR), and control without fertilizers (CK), same amount of N+P fertilizer was applied to the regimes except CK. High-throughput sequencing approaches were applied to the V3-V4 regions of the 16S ribosomal RNA for analysis of the bacterial abundance and community structures. Different long-term straw returning regimes significantly altered the physicochemical properties and bacterial communities of soil, among which CM had the most significant effects on soil fertility and bacterial diversity. Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes were consistently dominant in all soil samples, and Redundancy analysis (RDA) showed significant association of total nitrogen (TN), total phosphorus (TP) and available potassium (AK) with alternation of the bacterial community. Cattle manure had the most beneficial effects on soil fertility and bacterial diversity among different straw utilization and fertilization modes.