Heterologous Microarray Hybridization Between Vesicomyid Symbionts
ABSTRACT: The goal of this study was to use heterologous microarray hybridization to determine genomic content shared among different vesicomyid symbionts. These symbionts are closely related and can be thought of as different strains of bacteria, facilitating the use of heterologous microarray hybridization to determine genomic content. Keywords: comparative genomic hybridization Overall design: Microarrays were built off the Ruthia magnifica genome and two replicate hybridizations to this organism were used as a baseline for comparisons. Genomic DNA from two other vesicomyid symbionts (Calyptogena kilmeri and C. pacifica symbionts) was also hybridized to the array with three biological replicates for each sample.
Project description:The goal of this study was to use heterologous microarray hybridization to determine genomic content shared among different vesicomyid symbionts. These symbionts are closely related and can be thought of as different strains of bacteria, facilitating the use of heterologous microarray hybridization to determine genomic content. Keywords: comparative genomic hybridization Microarrays were built off the Ruthia magnifica genome and two replicate hybridizations to this organism were used as a baseline for comparisons. Genomic DNA from two other vesicomyid symbionts (Calyptogena kilmeri and C. pacifica symbionts) was also hybridized to the array with three biological replicates for each sample.
Project description:BACKGROUND: Hybridization of heterologous (non-specific) nucleic acids onto arrays designed for model-organisms has been proposed as a viable genomic resource for estimating sequence variation and gene expression in non-model organisms. However, conventional methods of normalization that assume equivalent distributions (such as quantile normalization) are inappropriate when applied to non-specific (heterologous) hybridization. We propose an algorithm for normalizing and centering intensity data from heterologous hybridization that makes no prior assumptions of distribution, reduces the false appearance of homology, and provides a way for researchers to confirm whether heterologous hybridization is suitable. RESULTS: Data are normalized by adjusting for Gibbs free energy binding, and centered by adjusting for the median of a common set of control probes assumed to be equivalently dissimilar for all species. This procedure was compared to existing approaches and found to be as successful as Loess normalization at detecting sequence variations (deletions) and even more successful than quantile normalization at reducing the accumulation of false positive probe matches between two related nematode species, Caenorhabditis elegans and C. briggsae. Despite the improvements, we still found that probe fluorescence intensity was too poorly correlated with sequence similarity to result in reliable detection of matching probe sequence. CONCLUSIONS: Cross-species hybridizations can be a way to adapt genome-enabled tools for closely related non-model organisms, but data must be appropriately normalized and centered in a way that accommodates hybridization of nucleic acids with diverged sequence. For short, 25-mer probes, hybridization intensity alone may be insufficiently correlated with sequence similarity to allow reliable inference of homology at the probe level.
Project description:The Vesicomyidae (Bivalvia: Mollusca) are a family of clams that form symbioses with chemosynthetic gamma-proteobacteria. They exist in environments such as hydrothermal vents and cold seeps and have a reduced gut and feeding groove, indicating a large dependence on their endosymbionts for nutrition. Recently, two vesicomyid symbiont genomes were sequenced, illuminating the possible nutritional contributions of the symbiont to the host and making genome-wide evolutionary analyses possible.To examine the genomic evolution of the vesicomyid symbionts, a comparative genomics framework, including the existing genomic data combined with heterologous microarray hybridization results, was used to analyze conserved gene content in four vesicomyid symbiont genomes. These four symbionts were chosen to include a broad phylogenetic sampling of the vesicomyid symbionts and represent distinct chemosynthetic environments: cold seeps and hydrothermal vents.The results of this comparative genomics analysis emphasize the importance of the symbionts' chemoautotrophic metabolism within their hosts. The fact that these symbionts appear to be metabolically capable autotrophs underscores the extent to which the host depends on them for nutrition and reveals the key to invertebrate colonization of these challenging environments.
Project description:BACKGROUND: Genome-wide analysis of sequence divergence among species offers profound insights into the evolutionary processes that shape lineages. When full-genome sequencing is not feasible for a broad comparative study, we propose the use of array-based comparative genomic hybridization (aCGH) in order to identify orthologous genes with high sequence divergence. Here we discuss experimental design, statistical power, success rate, sources of variation and potential confounding factors. We used a spotted PCR product microarray platform from Drosophila melanogaster to assess sequence divergence on a gene-by-gene basis in three fully sequenced heterologous species (D. sechellia, D. simulans, and D. yakuba). Because complete genome assemblies are available for these species this study presents a powerful test for the use of aCGH as a tool to measure sequence divergence. RESULTS: We found a consistent and linear relationship between hybridization ratio and sequence divergence of the sample to the platform species. At higher levels of sequence divergence (< 92% sequence identity to D. melanogaster) approximately 84% of features had significantly less hybridization to the array in the heterologous species than the platform species, and thus could be identified as "diverged". At lower levels of divergence (>or= 97% identity), only 13% of genes were identified as diverged. While approximately 40% of the variation in hybridization ratio can be accounted for by variation in sequence identity of the heterologous sample relative to D. melanogaster, other individual characteristics of the DNA sequences, such as GC content, also contribute to variation in hybridization ratio, as does technical variation. CONCLUSIONS: Here we demonstrate that aCGH can accurately be used as a proxy to estimate genome-wide divergence, thus providing an efficient way to evaluate how evolutionary processes and genomic architecture can shape species diversity in non-model systems. Given the increased number of species for which microarray platforms are available, comparative studies can be conducted for many interesting lineages in order to identify highly diverged genes that may be the target of natural selection.
Project description:BACKGROUND: Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available. RESULTS: We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested. CONCLUSIONS: MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions.
Project description:We describe the development and application of a Pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of S.aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty seven fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.
Project description:BACKGROUND: Unravelling the path from genotype to phenotype, as it is influenced by an organism's environment, is one of the central goals in biology. Gene expression profiling by means of microarrays has become very prominent in this endeavour, although resources exist only for relatively few model systems. As genomics has matured into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits. RESULTS: Here we present a microarray constructed with approximately 4500 features, derived from a brain-specific cDNA library for the African cichlid fish Astatotilapia burtoni (Perciformes). Heterologous hybridization, targeting RNA to an array constructed for a different species, is used for eight different fish species. We quantified the concordance in gene expression profiles across these species (number of genes and fold-changes). Although most robust when target RNA is derived from closely related species (<10 MA divergence time), our results showed consistent profiles for other closely related taxa (approximately 65 MA divergence time) and, to a lesser extent, even very distantly related species (>200 MA divergence time). CONCLUSION: This strategy overcomes some of the restrictions imposed on model systems that are of importance for evolutionary and ecological studies, but for which only limited sequence information is available. Our work validates the use of expression profiling for functional genomics within a comparative framework and provides a foundation for the molecular and cellular analysis of complex traits in a wide range of organisms.
Project description:Reef corals and sea anemones form symbioses with unicellular symbiotic dinoflagellates. The molecular circumventions that underlie the successful intracellular colonization of hosts by symbionts are still largely unknown. We conducted proteomic analyses to determine molecular differences of <i>Exaiptasia pallida</i> anemones colonized by physiologically different symbiont species, in comparison with symbiont-free (aposymbiotic) anemones. We compared one homologous species, <i>Symbiodinium linucheae</i>, that is natively associated with the clonal <i>Exaiptasia</i> strain (CC7) to another heterologous species, <i>Durusdinium trenchii</i>, a thermally tolerant species that colonizes numerous coral species. This approach allowed the discovery of a core set of host genes that are differentially regulated as a function of symbiosis regardless of symbiont species. The findings revealed that symbiont colonization at higher densities requires circumvention of the host cellular immunological response, enhancement of ammonium regulation, and suppression of phagocytosis after a host cell in colonized. Furthermore, the heterologous symbionts failed to duplicate the same level of homologous colonization within the host, evidenced by substantially lower symbiont densities. This reduced colonization of <i>D. trenchii</i> correlated with its inability to circumvent key host systems including autophagy-suppressing modulators, cytoskeletal alteration, and isomerase activity. The larger capability of host molecular circumvention by homologous symbionts could be the result of a longer evolutionary history of host/symbiont interactions, which translates into a more finely tuned symbiosis. These findings are of great importance within the context of the response of reef corals to climate change since it has been suggested that coral may acclimatize to ocean warming by changing their dominant symbiont species.
Project description:The objective of this work was to determine the effectiveness of cross-hybridization of gDNA from five native soil nematodes to an Affymetrix Caenorhabditis elegans tiling array. Cross-hybridization experiments using C. briggsae, for which genome information is available, allowed hybridisation intensities to be correlated with known sequence differences. Initial analysis of data by conventional array-based Comparative Genomic Hybridization (aCGH) techniques at the chip level lead to misleading results due to an artefact from the combination of scaling, bandwidth smoothing, and differential GC content in exon and intron regions. To circumvent this artefact, individual probes were instead normalized and centered by adjusting for probe-specific thermodynamic binding affinity. However, cross-hybridization of C. briggsae DNA revealed that the resultant probe intensities alone were still uncorrelated to sequence similarity below 90% identity. Below 90% similarity, all probes hybridize uniformly poorly, and above 90% similarity the hybridization differences are not large enough to detect over background, therefore, no 'threshold' ratio of hybridization intensity was successful at identifying probes with similarity to the heterologous genome. In light of the observations described here, we suggest that the criteria for replication and verification of gene expression profiles generated from cross-species microarray hybridizations be more stringent than typically adopted for con-specific hybridizations. Overall design: Genomic DNA from Caenorhabditis elegans N2 (Bristol), C. elegans CB4856 (Hawaiian), C. briggsae AF16, Oscheius tipulae KS585, Oscheius FVV-2 KS555, Mesorhabditis sp. KS587, Acrobeloides sp. KS586, and Chiloplacus sp. KS584 were hybridized onto C. elegans Affymetrix tiling array (two replicate chips were performed for each species).