A Transcriptome—Targeting EcoChip for Assessing Functional Mycodiversity
Ontology highlight
ABSTRACT:
ORGANISM(S): soil metagenome
PROVIDER: GSE28018 | GEO | 2011/11/14
SECONDARY ACCESSION(S): PRJNA137961
REPOSITORIES: GEO
Similar Datasets
Project description:A functional biodiversity microarray (EcoChip) prototype has been developed to facilitate the analysis of fungal communities in environmental samples with broad functional and phylogenetic coverage and to enable the incorporation of nucleic acid sequence data as they become available from large-scale (next generation) sequencing projects. A dual probe set (DPS) was designed to detect a) functional enzyme transcripts at conserved protein sites and b) phylogenetic barcoding transcripts at ITS regions present in precursor rRNA. Deviating from the concept of GeoChip-type microarrays, the presented EcoChip microarray phylogenetic information was obtained using a dedicated set of barcoding microarray probes, whereas functional gene expression was analyzed by conserved domain-specific probes. By unlinking these two target groups, the shortage of broad sequence information of functional enzyme-coding genes in environmental communities became less important. The novel EcoChip microarray could be successfully applied to identify specific degradation activities in environmental samples at considerably high phylogenetic resolution. Reproducible and unbiased microarray signals could be obtained with chemically labeled total RNA preparations, thus avoiding the use of enzymatic labeling steps. ITS precursor rRNA was detected for the first time in a microarray experiment, which confirms the applicability of the EcoChip concept to selectively quantify the transcriptionally active part of fungal communities at high phylogenetic resolution. In addition, the chosen microarray platform facilitates the conducting of experiments with high sample throughput in almost any molecular biology laboratory. In this study, two independent RNA samples from a pine forest soil were labelled and hybridised to a custom-made EcoChip microarray consisting of about 9000 probes targeting expressed fungals genes and about 5000 probes targeting the precursor-rRNA of different fungal lineages
2011-11-14 | E-GEOD-28018 | biostudies-arrayexpress
Project description:To effectively monitor microbial populations in acidic environments and bioleaching systems, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the known genes associated with the acidophiles. This array contained 1,072 probes in which there were 571 related to 16S rRNA and 501 related to functional genes. Acid mine drainage (AMD) presents numerous problems to the aquatic life and surrounding ecosystems. However, little is known about the geographic distribution, diversity, composition, structure and function of AMD microbial communities. In this study, we analyzed the geographic distribution of AMD microbial communities from twenty sites using restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, and the results showed that AMD microbial communities were geographically distributed and had high variations among different sites. Then an AMD-specific microarray was used to further analyze nine AMD microbial communities, and showed that those nine AMD microbial communities had high variations measured by the number of detected genes, overlapping genes between samples, unique genes, and diversity indices. Statistical analyses indicated that the concentrations of Fe, S, Ca, Mg, Zn, Cu and pH had strong impacts on both phylogenetic and functional diversity, composition, and structure of AMD microbial communities. This study provides insights into our understanding of the geographic distribution, diversity, composition, structure and functional potential of AMD microbial communities and key environmental factors shaping them. This study investigated the geographic distribution of Acid Mine Drainages microbial communities using a 16S rRNA gene-based RFLP method and the diversity, composition and structure of AMD microbial communities phylogenetically and functionally using an AMD-specific microarray which contained 1,072 probes ( 571 related to 16S rRNA and 501 related to functional genes). The functional genes in the microarray were involved in carbon metabolism (158), nitrogen metabolism (72), sulfur metabolism (39), iron metabolism (68), DNA replication and repair (97), metal-resistance (27), membrane-relate gene (16), transposon (13) and IST sequence (11).
2011-03-08 | E-GEOD-27542 | biostudies-arrayexpress
Project description:To effectively monitor microbial populations in acidic environments and bioleaching systems, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the known genes associated with the acidophiles. This array contained 1,072 probes in which there were 571 related to 16S rRNA and 501 related to functional genes. Acid mine drainage (AMD) presents numerous problems to the aquatic life and surrounding ecosystems. However, little is known about the geographic distribution, diversity, composition, structure and function of AMD microbial communities. In this study, we analyzed the geographic distribution of AMD microbial communities from twenty sites using restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, and the results showed that AMD microbial communities were geographically distributed and had high variations among different sites. Then an AMD-specific microarray was used to further analyze nine AMD microbial communities, and showed that those nine AMD microbial communities had high variations measured by the number of detected genes, overlapping genes between samples, unique genes, and diversity indices. Statistical analyses indicated that the concentrations of Fe, S, Ca, Mg, Zn, Cu and pH had strong impacts on both phylogenetic and functional diversity, composition, and structure of AMD microbial communities. This study provides insights into our understanding of the geographic distribution, diversity, composition, structure and functional potential of AMD microbial communities and key environmental factors shaping them.
2011-03-08 | GSE27542 | GEO
Project description:Despite the global importance of forests, it is virtually unknown how their soil microbial communities adapt at the phylogenetic and functional level to long term metal pollution. Studying twelve sites located along two distinct gradients of metal pollution in Southern Poland revealed that both community composition (via MiSeq Illumina sequencing of 16S rRNA genes) and functional gene potential (using GeoChip 4.2) were highly similar across the gradients despite drastically diverging metal contamination levels. Metal pollution level significantly impacted microbial community structure (p = 0.037), but not bacterial taxon richness. Metal pollution altered the relative abundance of specific bacterial taxa, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes and Proteobacteria. Also, a group of metal resistance genes showed significant correlations with metal concentrations in soil, although no clear impact of metal pollution levels on overall functional diversity and structure of microbial communities was observed. While screens of phylogenetic marker genes, such as 16S rRNA, provided only limited insight into resilience mechanisms, analysis of specific functional genes, e.g. involved in metal resistance, appeared to be a more promising strategy. This study showed that the effect of metal pollution on soil microbial communities was not straightforward, but could be filtered out from natural variation and habitat factors by multivariate statistical analysis and spatial sampling involving separate pollution gradients.
2014-07-22 | GSE59620 | GEO
Project description:Despite the global importance of forests, it is virtually unknown how their soil microbial communities adapt at the phylogenetic and functional level to long term metal pollution. Studying twelve sites located along two distinct gradients of metal pollution in Southern Poland revealed that both community composition (via MiSeq Illumina sequencing of 16S rRNA genes) and functional gene potential (using GeoChip 4.2) were highly similar across the gradients despite drastically diverging metal contamination levels. Metal pollution level significantly impacted microbial community structure (p = 0.037), but not bacterial taxon richness. Metal pollution altered the relative abundance of specific bacterial taxa, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes and Proteobacteria. Also, a group of metal resistance genes showed significant correlations with metal concentrations in soil, although no clear impact of metal pollution levels on overall functional diversity and structure of microbial communities was observed. While screens of phylogenetic marker genes, such as 16S rRNA, provided only limited insight into resilience mechanisms, analysis of specific functional genes, e.g. involved in metal resistance, appeared to be a more promising strategy. This study showed that the effect of metal pollution on soil microbial communities was not straightforward, but could be filtered out from natural variation and habitat factors by multivariate statistical analysis and spatial sampling involving separate pollution gradients. 12 samples were collected from two long-term polluted areas (Olkusz and Miasteczko M-EM-^ZlM-DM-^Eskie) in Southern Poland. In the study presented here, a consecutively operated, well-defined cohort of 50 NSCLC cases, followed up more than five years, was used to acquire expression profiles of a total of 8,644 unique genes, leading to the successful construction of supervised
2014-07-22 | E-GEOD-59620 | biostudies-arrayexpress
Project description:Microarrays are useful tools for detecting and quantifying specific functional and phylogenetic genes in natural microbial communities. In order to track uncultivated microbial genotypes and their close relatives in an environmental context, we designed and implemented a âgenome proxyâ microarray that targets microbial genome fragments recovered directly from the environment. Fragments consisted of sequenced clones from large-insert genomic libraries from microbial communities in Monterey Bay, the Hawaii Ocean Time-series station ALOHA, and Antarctic coastal waters. In a prototype array, we designed probe sets to thirteen of the sequenced genome fragments and to genomic regions of the cultivated cyanobacterium Prochlorococcus MED4. Each probe set consisted of multiple 70-mers, each targeting an individual ORF, and distributed along each ~40-160kbp contiguous genomic region. The targeted organisms or clones, and close relatives, were hybridized to the array both as pure DNA mixtures and as additions of cells to a background of coastal seawater. This prototype array correctly identified the presence or absence of the target organisms and their relatives in laboratory mixes, with negligible cross-hybridization to organisms having â¤~75% genomic identity. In addition, the array correctly identified target cells added to a background of environmental DNA, with a limit of detection of ~0.1% of the community, corresponding to ~10^3 cells/ml in these samples. Signal correlated to cell concentration with an R2 of 1.0 across six orders of magnitude. In addition the array could track a related strain (at 86% genomic identity to that targeted) with a linearity of R2=0.9999 and a limit of detection of ~1% of the community. Closely related genotypes were distinguishable by differing hybridization patterns across each probe set. This arrayâs multiple-probe, âgenome-proxyâ approach and consequent ability to track both target genotypes and their close relatives is important for the arrayâs environmental application given the recent discoveries of considerable intra-population diversity within marine microbial communities. Keywords: target addition experiment, proof-of-concept for GPL6012 ***Overall Array design*** The prototype microarray targeted thirteen BAC or fosmid genome fragments (20-160kb) from both bacteria and archaea, recovered from a variety of marine habitats, as well as the cyanobacterium Prochlorococcus MED4. These clones were originally sequenced because of the presence of taxonomic marker or specific functional genes. This array consisted of sets of 70-bp oligonucleotides targeting each genome or genome fragment (Fig. 1), dispersed along the target sequences with no more than one probe per gene, and excluding rRNA genes as targets. The probes were selected solely based on theoretical thermodynamic properties and GC content (~40%); that is, probe selection did not focus on specific genes or regions, but simply produced the âoptimalâ probes for each genome proxy based on the probesâ predicted hybridization properties. rRNA genes were excluded, because this probe design approach, which avoids sequence alignments and considerations of RNA secondary structure, would be unlikely to result in useful rRNA probes. Furthermore, rRNA probes of traditional design could not be included on the array because their appropriate hybridization conditions would be very different from those of this arrayâs probes. ***Microarray probe design*** Microarray 70-mer probes were designed using the program ArrayOligoSelector (Zhu et al., 2003) with the following settings: target %GC = 40%, 1 probe/gene, with the ORFs for each genome fragment as both the input and the database file. The output candidate 70mers were then sorted based on their %GC and those closest to 40% were chosen. In the case of more than the target number of probes having 40%GC, the subset with the lowest free energy of hybridization were selected as probes. Generally, 20 probes were selected per organism. Prochlorococcus MED4 was represented by 60 probes total, 20 each for three different 80kb âgenome-proxyâ regions: 0-80kb, 1.29-1.37Mbp, and 1.58 to 1.66Mbp. Using the same method, a set (n=20) of positive control probes were designed to the genome of the halophillic archaeon Halobacterium salinarum NRC-1. Negative control probes (n=28) were designed to a set of 49 random 1000-base sequences (Stothard, 2000). ***Microarray construction and hybridization*** Oligonucleotides were synthesized (Illumina, San Diego, California), suspended in 3XSSC to a concentration of 40pmol/μl, and spotted on homemade poly-L-lysine-coated glass slides using a QArray 2 microarraying robot (Genetix, Hampshire, England). Six replicates of each probe were spotted. ***Microarray data analysis*** Hybridized arrays were scanned using an Axon Instruments 4000B scanner (Foster City, CA) and the data was normalized and filtered using perl scripts written for the purpose, by the following steps. (1) Signal intensities for each spot were calculated by subtracting the local background (mean F532 â median B532, as calculated by GenePix Pro 5.1 software, Axon Instruments). (2) The median value across replicates was calculated for each probe. (3) For each probe set, the number of probes greater than twice the mean negative control signal was calculated, before further processing. (4) Filter I: Arrays with less than half their positive control probes exceeding twice the mean negative control signal were considered poor quality, low dynamic range, arrays and were excluded from further analysis. (5) Each probe signal was corrected for non-specific binding by subtracting the mean negative control spot signal. (6) The data was then normalized for array-to-array variations in brightness by dividing each probe signal by the mean positive control signal. This positive control signal was the mean signal across the Halobacterium salinarum probes in each hybridization, with identical amounts of H. salinarum DNA having been added to each reaction prior to amplification and labeling. (7) Filter II: In order for a genotype to be considered âpresentâ, at least 45% of its probes had to exceed twice the mean negative control signal. (8) Finally, each genotype signal was calculated as either the MEAN or TUKEY BIWEIGHT across its probe set. ***Experimental Design*** The array was hybridized to laboratory mixtures of cloned environmental genomic DNA targeted by the array, in varying ratios. The use of multiple probes to target many genes from each organism helped to normalize probe-to-probe heterogeneity, by averaging across all probes in a set (as described below). The evenness of probe response across each genotypeâs set was also used to evaluate the relatedness of hybridizing DNA. To more precisely define the arrayâs phylogenetic range and specificity, it was tested against DNA from Prochlorococcus MED4 and related strains, spanning the known range of Prochlorococcus phylogenetic diversity. To test the effects of hybridization stringency on the specificity and signal of the MED4 probes, Prochlorococcus strains were hybridized at a range of conditions. To test whether the specificity results for Prochlorococcus were comparable for other targeted clades, two genome fragments recovered from closely related phylotypes within the SAR86 clade of the gamma-proteobacteria were represented on the array, and were tested for specificity. To understand the equivalence of probe sets targeting different regions of the same organismâs genome, we targeted three 80kb âgenome proxyâ regions of the Prochlorococcus MED4 genome. One of the regions fell in a genomic âislandâ where inter-strain variability is concentrated (âISL5â in Coleman et al., 2006). To test the array in a complex environmental context, we collected coastal seawater (lacking detectable Prochlorococcus cells by flow cytometry) and added Prochlorococcus cells from strains MED4, MIT9515, MIT9312 and MIT9313 over a range of concentrations from ~101 â 106 cells/ml (Fig. 3). The seawater was then filtered and the DNA extracted, amplified, labeled, and hybridized to the array.
2010-05-25 | E-GEOD-9384 | biostudies-arrayexpress
Project description:Waste decomposition in landfills is a complex and microbe-mediated process. Understanding the microbial community composition and structure is critical for accelerating decomposition and reducing adverse impact on the environment. Here, we examined the microbial communities along with landfill depth and age (LDA) in a sanitary landfill in Beijing, China using 16s rRNA Illumina sequencing and GeoChip 4.6. We found that Clostridiales and Methanofollis were the predominant bacteria and archaea in the present landfill, respectively. Interestingly, in contrast with the decreasing trend of microbial diversity in soil, both phylogenetic and functional diversities were higher in deeper and older refuse in the landfill. Phylogenetic compositions were obviously different in the refuse with the same LDA and such difference is mainly attributed to the heterogeneity of refuse instead of random process. Nevertheless, functional structures were similar within the same LDA, indicating that microbial community assembly in the landfill may be better reflected by functional genes rather than phylogenetic identity. Mantel test and canonical correspondence analysis suggested that environmental variables had significant impacts on both phylogenetic composition and functional structure. Higher stress genes, genes for degrading toxic substances and endemic genes in deeper and older refuse indicated that they were needed for the microorganisms to survive in the more severe environments. This study suggests that landfills are a repository of stress-resistant and contaminant-degrading microorganisms, which can be used for accelerating landfill stabilization and enhancing in situ degradation. Fifteen refuse samples with five landfill depths and ages (6m/2a, 12m/4a, 18m/6a, 24m/8a and 30m/10a) were collected from a sanitary landfill in Beijing, China. Three replicates in every landfill depth and age
2016-06-15 | E-GEOD-68712 | biostudies-arrayexpress
Project description:Microbial community analysis with DNA oligonucleotide microarrays targeting ribosomal RNA (rRNA) provides a highly parallel interrogation of nucleic acids isolated from environmental samples. High fidelity readout is essential for accurate interpretation of hybridisations. We describe the hybridisation of in vitro transcribed 16S rRNA from an uncontaminated and 2,4,6-trinitrotoluene contaminated soil to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and their 2 corresponding mismatch (MM) probes. Thermal dissociation analysis was used to determine the specificity of each PM-MM probe set. Functional ANOVA often discriminated PM-MM probe sets when Td values (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greater than the Td. Comparison of signal intensities measured prior to dissociation analysis from hybridisations of the two soil samples revealed significant differences in domain-, group- and species-specific probes. Functional ANOVA showed significantly different dissociation curves for 11 PM probes when hybridisations from the two soil samples were compared, even though initial signal intensities for 3 of the 11 did not vary. This approach provides a highly parallel, multi-level analysis that incorporates MM probes and dissociation curves into high fidelity microarray analysis of complex environmental nucleic acid profiles. Keywords: Microbial diversity, thermal dissociation analysis
2005-10-26 | GSE3499 | GEO
Project description:Understanding the bacterial community structure, and their functional analysis for active bioremediation process is essential to design better and cost effective strategies. Microarray analysis enables us to simultaneously study the functional and phylogenetic markers of hundreds of microorganisms which are involved in active bioremediation process in an environment. We have previously described development of a hybrid 60-mer multibacterial microarray platform (BiodegPhyloChip) for profiling the bacterial communities and functional genes simultaneously in environments undergoing active bioremediation process (Pathak et al; Appl Microbiol Biotechnol,Vol. 90, 1739-1754). The present study involved profiling the status of bacterial communities and functional (biodegradation) genes using the developed 60-mer oligonucleotide microarray BiodegPhyloChip at five contaminated hotspots in the state of Gujarat, in western India. The expression pattern of functional genes (coding for key enzymes in active bioremediation process) at these sites was studied to understand the dynamics of biodegradation in the presence of diverse group of chemicals. The results indicated that the nature of pollutants and their abundance greatly influence the structure of bacterial communities and the extent of expression of genes involved in various biodegradation pathways. In addition, site specific factors also play a pivotal role to affect the microbial community structure as was evident from results of 16S rRNA gene profiling of the five contaminated sites, where the community structure varied from one site to another drastically.
2012-12-01 | GSE38004 | GEO
Project description:Waste decomposition in landfills is a complex and microbe-mediated process. Understanding the microbial community composition and structure is critical for accelerating decomposition and reducing adverse impact on the environment. Here, we examined the microbial communities along with landfill depth and age (LDA) in a sanitary landfill in Beijing, China using 16s rRNA Illumina sequencing and GeoChip 4.6. We found that Clostridiales and Methanofollis were the predominant bacteria and archaea in the present landfill, respectively. Interestingly, in contrast with the decreasing trend of microbial diversity in soil, both phylogenetic and functional diversities were higher in deeper and older refuse in the landfill. Phylogenetic compositions were obviously different in the refuse with the same LDA and such difference is mainly attributed to the heterogeneity of refuse instead of random process. Nevertheless, functional structures were similar within the same LDA, indicating that microbial community assembly in the landfill may be better reflected by functional genes rather than phylogenetic identity. Mantel test and canonical correspondence analysis suggested that environmental variables had significant impacts on both phylogenetic composition and functional structure. Higher stress genes, genes for degrading toxic substances and endemic genes in deeper and older refuse indicated that they were needed for the microorganisms to survive in the more severe environments. This study suggests that landfills are a repository of stress-resistant and contaminant-degrading microorganisms, which can be used for accelerating landfill stabilization and enhancing in situ degradation.
2016-06-15 | GSE68712 | GEO