New resources for functional analysis of omics data for the genus Aspergillus
© Nitsche et al; licensee BioMed Central Ltd. 2011
Received: 4 July 2011
Accepted: 5 October 2011
Published: 5 October 2011
Detailed and comprehensive genome annotation can be considered a prerequisite for effective analysis and interpretation of omics data. As such, Gene Ontology (GO) annotation has become a well accepted framework for functional annotation. The genus Aspergillus comprises fungal species that are important model organisms, plant and human pathogens as well as industrial workhorses. However, GO annotation based on both computational predictions and extended manual curation has so far only been available for one of its species, namely A. nidulans.
Based on protein homology, we mapped 97% of the 3,498 GO annotated A. nidulans genes to at least one of seven other Aspergillus species: A. niger, A. fumigatus, A. flavus, A. clavatus, A. terreus, A. oryzae and Neosartorya fischeri. GO annotation files compatible with diverse publicly available tools have been generated and deposited online. To further improve their accessibility, we developed a web application for GO enrichment analysis named FetGOat and integrated GO annotations for all Aspergillus species with public genome sequences. Both the annotation files and the web application FetGOat are accessible via the Broad Institute's website (http://www.broadinstitute.org/fetgoat/index.html). To demonstrate the value of those new resources for functional analysis of omics data for the genus Aspergillus, we performed two case studies analyzing microarray data recently published for A. nidulans, A. niger and A. oryzae.
We mapped A. nidulans GO annotation to seven other Aspergilli. By depositing the newly mapped GO annotation online as well as integrating it into the web tool FetGOat, we provide new, valuable and easily accessible resources for omics data analysis and interpretation for the genus Aspergillus. Furthermore, we have given a general example of how a well annotated genome can help improving GO annotation of related species to subsequently facilitate the interpretation of omics data.
Gene Ontology (GO) is a framework for functional annotation of gene products aiming to provide a unique vocabulary for living systems . It comprises Biological Process (BP), Molecular Function (MF) and Cellular Component (CC) ontologies. GO terms are organized as directed acyclic graphs (DAG) meaning that GO terms are connected as nodes by directed edges defining hierarchical parent-child relationships. As a consequence, the specificity of GO terms increases with increasing distance from their root node. Enrichment analysis of GO terms is a well accepted approach to dissecting omics data in a non-biased manner. It has been used in many studies to highlight major trends in genomic, transcriptomic or proteomic datasets and describe them with a controlled vocabulary [2–5]. If the frequency of specific GO terms in a list of genes or proteins is higher than expected by chance, it is likely that these enriched GO terms are related to the biological processes under investigation.
The genus Aspergillus covers a group of filamentous fungi that includes saprophytes, human and plant pathogens as well as species being exploited in biotechnology. Whereas A. nidulans has been comprehensively studied and used as model organism, A. niger, A. oryzae and A. terreus are important industrial workhorses for the production of various enzymes and organic acids. In medical research, A. fumigatus and Neosartorya fischeri are intensively studied because of their importance as allergens and pathogens of immunocompromised patients. The aflatoxin producing fungus A. flavus is well known to cause spoilage of a great variety of agricultural goods. With genome sequences publicly available for eight of its species, the genus Aspergillus provides an important group of related fungal species for comparative genomics . The exceptional role of this genus in the genomics of filamentous fungi is further emphasized by a community sequencing project (CSP#350), which has recently been initiated by the DOE Joint Genome Institute (JGI), aiming to sequence nine additional Aspergillus species. However, despite the importance of the genus Aspergillus, A. nidulans has so far been the only species with a genome-scale GO annotation inferred from both orthology mapping and intense manual curation [7–9], thus providing a valuable resource for the analysis of omics data.
In this work, we have generated a new central repository for functional analysis of omics data for the genus Aspergillus using GO annotation. Firstly, we extended the GO annotation of A. nidulans to all Aspergillus species with publicly available genome sequences and generated annotation files compatible with diverse publicly available tools for GO enrichment analysis. Secondly, we further improved the accessibility of the GO annotation for the genus Aspergillus by integrating it into a web tool for GO enrichment analysis and graph visualization named Fisher's exact test Gene Ontology annotation tool (FetGOat). Finally, we performed two case studies to demonstrate the value and flexibility of the newly generated resources for functional analysis of omics data for the genus Aspergillus.
Mapping of GO annotation
A. nidulans is the only Aspergillus species for which comprehensive GO annotation based on both computational prediction and extended manual curation of gene-specific literature is available . It constitutes a valuable resource for GO enrichment analysis, which has proven to be a powerful tool for dissecting omics data, for example sets of differentially expressed genes. The GO annotation of A. nidulans available at the Aspergillus Genome Database (AspGD)  covers 33% (3,498) of its predicted transcripts and associates them with 3,340 GO terms. Including all parental nodes, the list of GO terms extends to 5,508 comprising 3,061 (55%) BP, 1,753 MF (32%) and 694 (13%) CC terms.
Mapping of A. nidulans GO annotation
GO annotated (%)
Availability of GO resources for the genus Aspergillus
The newly mapped GO annotations were deposited at the Broad Institute's website (http://www.broadinstitute.org/fetgoat/index.html). Different annotation file formats were generated that can be used with diverse public tools for GO enrichment analysis, such as: the Gene Set Enrichment Analysis tool (GSEA) , the functional annotation suite Blast2GO , the Cytoscape plug-in BiNGO  and the Bioconductor package TopGO . To further improve its accessibility, we have implemented Fisher's exact test , a well-accepted approach for GO enrichment analysis, in the web application FetGOat and integrated the newly mapped GO annotations. FetGOat can be accessed via a web interface at the Broad Institute's website (http://www.broadinstitute.org/fetgoat/index.html). It combines GO annotations for all Aspergillus species with public genome sequences and a widely used statistical methodology to identify overrepresented GO terms. Via the web interface, a list of gene identifiers can be uploaded to the server and statistical parameters can easily be adjusted with end-user computational skills. After completion of the analysis on the server-side, the enrichment results are sent by Email. The results consist of plain text and spreadsheet files as well as scalable vector graphics representing graphs of enriched GO terms.
To demonstrate the flexibility and value of the newly generated resources for omics data analysis, we performed two case studies analyzing transcriptomic datasets recently published for the genus Aspergillus. In the first case study, we demonstrate that the generated resources can be used with various methods for enrichment analysis. We analyze a set of maltose-induced genes from A. niger using FetGOat and two alternative tools for enrichment analysis to subsequently compare their results. In the second case study, we highlight the advantage of having GO annotations that are as comprehensive as possible available for different species. We use FetGOat to analyze sets of glycerol-induced genes derived from a three-species microarray study to highlight major differences in the transcriptional responses for A. nidulans, A. niger and A. oryzae.
The first dataset reflects the transcriptomic responses of A. niger to growth in maltose and xylose-limited chemostat cultures at identical growth rates. From manual analysis of roughly 700 upregulated genes, Jørgensen et al.  concluded a concerted induction of secretory pathway genes in maltose compared to xylose-limited cultures.
FetGOat enrichment analysis of maltose-induced genes
Translocation to ER
posttranslational protein targeting
signal peptidase complex
SRP-dependent cotranslational protein targeting
protein maturation by peptide bond cleavage
Glycosylation in ER
endoplasmic reticulum lumen
protein amino acid N-linked glycosylation
transferase activity, transferring hexosyl groups
Transport between ER and golgi
COPI vesicle coat
COPII vesicle coat
ER to Golgi vesicle-mediated transport
integral to Golgi membrane
starch metabolic process
alcohol metabolic process
protein disulfide isomerase activity
acetate metabolic process
gamma-aminobutyric acid transport
small ribosomal subunit
For comparison of FetGOat with alternative programs, we used the generated annotation files and repeated the enrichment analysis with two publicly available tools, Blast2GO  and GSEA . The numbers of enriched GO terms found with Blast2GO and GSEA are in the same range compared to the results from FetGOat, they identified 76 and 47 enriched GO terms, respectively. To compare the enrichment results from the three tools, we computed semantic similarity scores with the G-SESAME tool . For both FetGOat and Blast2GO, the enrichment statistic is based on Fisher's exact test and thus their results are theoretically expected to be identical resulting in a semantic similarity score of 1. A similarity score of 0.983 confirms that their results are virtually identical, with minor differences that are likely due to differences in their implementations. In contrast to FetGOat (and Blast2GO), the GSEA results are based on running-sum statistics computed from the complete expression data set. Therefore, the similarity between their results can be expected to be less. Accordingly, G-SESAME determined a smaller semantic similarity score of 0.863 for the results obtained with FetGOat and the GSEA tool.
In addition to the GO terms identified by both Fisher's exact test based tools, GSEA computed an enrichment of GO terms related to oxidative phosphorylation (GO:0006119), carbohydrate transport (GO:0008643) and glucosidase activity (GO:0015926). Comparing maltose to xylose limitation, an enrichment of those GO terms fits our expectations. Under maltose-limitation, A. niger breaks down the disaccharide into its monomer glucose by enzymes having glucosidase activity. Subsequently, glucose is taken up by carbohydrate transporters, which can be expected to be different from those required for the uptake of xylose. Finally, 1 mole of glucose yields more ATP than 1 mole of xylose, thereby explaining an induction of oxidative phosphorylation.
These differences in the enrichment results are potentially inherited by the statistics applied by Jørgensen et al. to define the set of maltose-induced genes. In contrast to the GSEA tool, which analyzes the complete expression data, FetGOat and Blast2GO are depending on a-priori performed statistics that were applied to generate subsets of genes or proteins of interest. Jørgensen et al. used the Affymetrix MAS 5.0 algorithm for data pre-processing in combination with the student's t-test to define their set of maltose induced genes. In current literature, this approach is critically discussed [18, 19]. To assess the effect of those a-priori applied statistics on the differences between the results from FetGOat and the GSEA tool, we generated an alternative set of maltose-induced genes. We computed RMA expression data  from the raw data (CEL files) and subsequently applied a moderated t-statistic  to identify upregulated genes (data not shown). Interestingly, FetGOat also identified enriched GO terms related to glucosidase activity and carbohydrate transport for this alternative set of maltose-induced genes. However, no enrichment of genes related to oxidative phosphorylation was found. Genes annotated with the GO term oxidative phosphorylation were only marginally induced and their FDR values were rather high (data not shown). Interestingly, similar differences between Fisher's exact test based methods and the GSEA tool were reported in another study. In muscle tissue from diabetics, the GSEA tool identified a joint downregulation of genes related to oxidative phosphorylation compared to healthy controls, while no enrichment was found in the set of downregulated genes . For tightly regulated essential cellular processes that show only minor fold changes, the GSEA tool seems to be superior to gene-by-gene differential expression studies.
In the second case study, we used FetGOat to analyze transcriptomic data generated by Salazar et al. . With a three-species microarray, the authors studied the transcriptomic responses of A. nidulans, A. niger and A. oryzae to growth in glycerol and glucose-limited batch cultures. The authors identified 4,139 glycerol-induced genes comprising 679, 2,240 and 1,040 genes from A. nidulans, A. niger and A. oryzae, respectively. Based on tri-directional best blast hits, 81 orthologous gene clusters were shown to be upregulated in each of the species. Using the A. niger (strain ATCC 1015) GO annotation, Salazar et al. analyzed the set of conserved upregulated genes and identified enriched BP terms, which are related to amino acid metabolism, gluconeogenesis, hexose and alcohol biosynthetic processes.
Corresponding to the enrichment results from Salazar et al., FetGOat identified enriched GO terms that are related to pyruvate and (aromatic) amino acid metabolism. Unlike Salazar et al., FetGOat did not identify BP terms related to gluconeogenesis. This difference can be explained by an improvement of the GO annotation. While only three genes were annotated with the BP term gluconeogenesis (GO:0006094) in the GO annotation used by Salazar et al., it is a total of 28 genes in the newly mapped GO annotation for A. niger (ATCC 1015 strain). For both annotations, one out of the upregulated conserved genes is annotated by the BP term gluconeogenesis, thus explaining why Salazar et al. identified it as an enriched BP term and FetGOat did not.
A detailed and comprehensive genome annotation can be considered a prerequisite for the analysis and interpretation of omics data. GO provides a framework for functional annotation and has been proven to be a valuable tool for omics data analysis, especially in combination with enrichment statistics. Currently, the GO reference genome project  provides the most comprehensive manually curated GO annotation for twelve model organisms and is intended to serve as a reference for automated mapping of GO annotation to organisms other than these major models. From the reference genome projects, Saccharomyces cerevisiae and Schizosaccharomyces pombe are most closely related to the genus Aspergillus.
A. nidulans has so far been the only Aspergillus species with comprehensive genome scale GO annotation based on both orthology mapping to S. cerevisiae and extensive manual curation  of gene-specific literature. We have thus mapped the A. nidulans GO annotation to all other Aspergillus species (see Table 1) with published genomes. With 79% of all A. nidulans genes being organized in Jaccard orthologous clusters covering 97% of all its GO annotated genes, we demonstrated that this approach is promising for mapping GO annotation between closely related genomes such as those of the genus Aspergillus. Nevertheless, the newly generated GO annotations have exclusively been inferred by computational analysis and thus their quality can be expected to be lower compared to the extensively manually curated A. nidulans GO annotation. The ortholog clustering approach as implemented in the Sybil comparative analysis package  has worked well for a number of comparative genome studies [24–33], but does have limitations, especially when there are a large number of strains and/or percentage of repetitive proteins. Additionally, we recognize that the optimal choice of an ortholog detection method depends on the purpose of the analysis. This graph based approach is robust if looking at closely related species, but may not be the best choice when considering large numbers of more distantly related genomes.
The GO annotations for ten Aspergillus strains (see Table 1) have been made available at the Broad Institute's website (http://www.broadinstitute.org/fetgoat/index.html) and will be updated regularly as the GO annotations for the various Aspergillus species continue to improve through manual and computational efforts. To improve the applicability of the GO annotations, they are provided in different file formats that can be used with various freely available GO enrichment tools, e.g. Blast2GO , TopGO , GSEA  and BinGO . Thereby, functional analysis of Aspergillus omics data by GO enrichment analysis is strongly facilitated. The availability of different annotation file formats makes it feasible to use different tools and compare them with each other.
To further improve the accessibility of the extended annotations, we developed the web application FetGOat and integrated the GO annotation for all Aspergillus species with public genome sequences. FetGOat basically resembles the functionality of other publicly available enrichment tools. However, for the Aspergillus research community, FetGOat is a valuable addition to existing programs because it uniquely combines an intuitive web interface, GO annotations for all Aspergilli with public genome sequences and a frequently applied statistical method for the identification of enriched GO terms.
To demonstrate the use of those newly generated resources for functional analysis of omics data, we applied them in two case studies to re-analyze recently published microarray data in an automated and un-biased manner. As shown for the first dataset, the enrichment results are in correspondence to the main conclusions from Jørgensen et al. . We found an induction of processes related to secretion, glycosylation and starch degradation (see Table 2). In addition, we used the dataset from Jørgensen et al. to compare the enrichment results of FetGOat to those obtained with two well established publicly available tools, Blast2GO and GSEA. The three tools apply two different methods for enrichment analysis. While Blast2GO and FetGOat compute a Fisher's exact test statistic to identify GO terms that are over-represented in subsets of genes derived e.g. from transcriptomic or proteomic data, the GSEA tool computes running sum statistics on (non-filtered) expression data to identify a-priori defined groups of genes that show joined differential expression. The results from FetGOat are virtually identical to the results obtained with Blast2GO demonstrating the correctness of FetGOat. As expected, the similarity between the results from FetGOat and the GSEA tool is less, while their results are still well comparable. For a large part, both tools are highlighting the same transcriptional trends. However, the GO term oxidative phosphorylation was exclusively identified as being enriched by the GSEA tool. Taking into account that 1 mole of glucose yields more ATP than 1 mole of xylose, an induction of the oxidative phosphorylation machinery during growth in maltose-limited cultures can be expected. Because the fold-changes of the corresponding genes were very small and their statistical significances were low, no enrichment could be found in the set of maltose-induced genes as assessed by Fisher's exact test. Similar results were found in another study, in which the GSEA tool detected a joined transcriptional downregulation of genes related to oxidative phosphorylation in tissue from diabetics vs. control . For tightly regulated essential genes, which show only marginal differential expression, the GSEA tool seems to be superior to gene-by-gene differential expression approaches. However, we would like to emphasize that this is rather caused by the a-priori performed statistics than by the Fisher's exact test itself. A combination of clustering based on gene expression profiles combined with Fisher's exact test enrichment statistics will potentially allow to draw similar conclusions as with the GSEA tool. The causality between an increased ATP yield for maltose and an upregulation of secretion related genes remains to be investigated. However, it is an interesting new hypothesis for further investigations.
For the second dataset from Salazar et al. , we first performed GO enrichment analysis on the set of 81 conserved and glycerol-induced genes used in the original study. We could partly reproduce the enrichment results. However, we didn't find an enrichment of genes annotated with the GO term gluconeogenesis. A comparison of the GO annotation used by Salazar et al. and our newly mapped GO annotation revealed that this is due to an improvement of the newly mapped GO annotation, which includes many more genes annotated with the GO term gluconeogenesis. As expected from analyzing orthologous gene sets, we showed that the enrichment results are nearly identical, independent of which of the three Aspergilli they were obtained for. Furthermore, we separately performed enrichment analysis for the three Aspergilli analyzing their complete sets of up regulated genes and highlighted major differences in their responses to glycerol vs. glucose limitation. Thereby, we were able to draw additional conclusions explaining their different capabilities to grow on glycerol. Especially for the three-species microarray platform, FetGOat in combination with the newly mapped GO annotation forms a new, valuable and flexible resource for omics data analysis. Applied at an early stage of data analysis, GO enrichment analysis can thus strongly facilitate subsequent manual data interpretation.
While GSEA is an attractive alternative to Fisher's exact test based tools such as FetGOat and Blast2GO, it lacks flexibility because it is restricted to transcriptomic data and can only compare two conditions at a time. Furthermore, its application is more sophisticated, because microarray specific chip annotation files as well as phenotypic labels have to be provided for analysis. Tools such as FetGOat and Blast2GO can be applied to any set of genes or proteins deriving from genomic, transcriptomic or proteomic studies. They can for example be used to perform GO enrichment analysis on a set of proteins commonly secreted under certain conditions. Improving the power of the statistics applied to obtain gene sets of interest will consequently improve the strength of Fisher's exact test based enrichment analysis. For transcriptomic data analysis, moderated statistics or non specific filtering have for example been shown to improve the statistical power .
The choice of a tool for GO enrichment analysis depends on the type of data, the available resources and personal preferences. Certainly, most of the enrichment results will be redundant between the tools. With the different GO annotation files generated in this study, various freely available tools can easily be used and compared with each other. Especially for the genus Aspergillus, FetGOat stands out with respect to the ease of use and the integration of comprehensive and regularly updated GO annotations. The power of FetGOat lies in its flexibility. Any set of genes/proteins from any Aspergillus strain with published genome sequence can be investigated for enrichment of GO terms. FetGOat is not restricted to the genus Aspergillus as it can be extended to include GO annotations from any organism of interest.
We have mapped the A. nidulans GO annotation to the genomes of seven other Aspergillus species and made the GO annotations available in different file formats. We furthermore developed the web tool FetGOat, which can be used for GO enrichment analysis of omics data from all Aspergillus strains with published genome sequences. Both, the mapped GO annotations and FetGOat were successfully applied in two case studies and are available at the Broad Institute's website (http://www.broadinstitute.org/fetgoat/index.html). Moreover, we have given a general example of how a well annotated genome can help improving GO annotation of related species to subsequently facilitate the interpretation of omics data.
Ortholog and paralog identification
Using default parameters, any pair of polypeptides with a Jaccard coefficient > 0.6 is connected in a graph representation. The connected components of this graph are referred to as "Jaccard Clusters" and are analogous to paralogous protein clusters within each species. Subsequently, the reciprocal best-hit phase of the clustering algorithm identifies pairs of Jaccard clusters such that: (1) The clusters are from different genomes. (2) The highest-scoring BLASTP match of at least one polypeptide in each of the clusters is to a polypeptide in the other cluster. A graph is constructed, with an edge drawn between two nodes (Jaccard clusters) if and only if they are bidirectional best BLASTP matches of each other. The connected components of this graph are considered ortholog groups in downstream analysis and will be referred to as "Jaccard orthologous clusters".
Mapping A. nidulans GO annotation
GO annotation for A. nidulans (gene_association.aspgd version: 1.256) was obtained from the Aspergillus Genome Database (AspGD: http://www.aspgd.org)  and is based on orthology mapping between A. nidulans and S. cerevisiae as well as extensive manual curation based on gene specific A. nidulans literature. GO terms for Jaccard orthologous clusters and their associated proteins were inferred from A. nidulans GO annotation such that each protein belonging to the same Jaccard orthologous cluster shares identical GO terms. For each of the analyzed strains (see Table 1), individual GO annotation files were generated in different formats.
GO enrichment analyses were performed applying two different statistical tests: Fisher's exact test  and Kolmogorow-Smirnov statistics [11, 35]. If not stated differently, p-values were corrected according to Benjamini & Hochberg  and a critical False Discovery Rate (FDR) q-value of 0.05 was applied. For the Fisher's exact test based enrichment analysis of GO terms, we developed the web application FetGOat, which calculates one-tailed p-values and corrects them for multiple hypothesis testing according to the Benjamini & Hochberg method. In addition to FetGOat, Blast2GO  was used to compute enriched GO terms via Fisher's exact test as implemented in GOSSIP . For the identification of enriched GO terms based on the Kolmogorov-Smirnov statistic, the GSEA tool  was used. The corresponding GO annotation files for Blast2GO and the GSEA tool were generated in this study and are available at the Broad Institute's website (http://www.broadinstitute.org/fetgoat/index.html).
Mapping to GO slim annotation
To summarize GO enrichment results, we mapped the enriched GO terms to a GO Slim annotation , which is a reduced version of the complete annotation with less detailed high-level GO terms, and counted the occurrences (single occurrence option) of GO Slim terms as well as related lower hierarchy terms using the CateGOrizer tool .
This work was supported by grants of the SenterNovem IOP Genomics project (IGE07008) and the National Institute of Allergy and Infectious Diseases at the US National Institutes of Health (R01 AI077599). Part of this work was carried out within the research programme of the Kluyver Centre for Genomics of Industrial Fermentation, which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research.
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