- Open Access
Quail Genomics: a knowledgebase for Northern bobwhite
© Perkins et al; licensee BioMed Central Ltd. 2010
Published: 7 October 2010
The Quail Genomics knowledgebase (http://www.quailgenomics.info) has been initiated to share and develop functional genomic data for Northern bobwhite (Colinus virginianus). This web-based platform has been designed to allow researchers to perform analysis and curate genomic information for this non-model species that has little supporting information in GenBank.
A multi-tissue, normalized cDNA library generated for Northern bobwhite was sequenced using 454 Life Sciences next generation sequencing. The Quail Genomics knowledgebase represents the 478,142 raw ESTs generated from the sequencing effort in addition to assembled nucleotide and protein sequences including 21,980 unigenes annotated with meta-data. A normalized MySQL relational database was established to provide comprehensive search parameters where meta-data can be retrieved using functional and structural information annotation such as gene name, pathways and protein domain. Additionally, blast hit cutoff levels and microarray expression data are available for batch searches. A Gene Ontology (GO) browser from Amigo is locally hosted providing 8,825 unigenes that are putative orthologs to chicken genes. In an effort to address over abundance of Northern bobwhite unigenes (71,384) caused by non-overlapping contigs and singletons, we have built a pipeline that generates scaffolds/supercontigs by aligning partial sequence fragments against the indexed protein database of chicken to build longer sequences that can be visualized in a web browser.
Our effort provides a central repository for storage and a platform for functional interrogation of the Northern bobwhite sequences providing comprehensive GO annotations, meta-data and a scaffold building pipeline. The Quail Genomics knowledgebase will be integrated with Japanese quail (Coturnix coturnix) data in future builds and incorporate a broader platform for these avian species.
Northern bobwhite represents an avian wildlife species that is critical to the maintenance of ecosystem function. The munitions compound (MC) 2,6-dinitrotoluene (2,6-DNT) contaminates soil and water on military training facilities posing a hazard to Northern bobwhite, a ground foraging bird. Advancing our knowledge in the potential hazards of 2,6-DNT to avian species is important to insure the protection of these wild populations. The utility of Northern bobwhite in various ecological , experimental  and regulatory  scenarios has gained recognition for the species as an excellent experimental avian wildlife model.
Genomic information for Northern bobwhite lags behind other avian model species with 1717 ESTs derived from a brain-tissue cDNA library . This low coverage of the Northern bobwhite genome limits application of toxicogenomic approaches for assessing the systemic impacts of chemical stressors. In comparison, avian species such as chicken and zebra finch have been robustly described and microarrays have been developed to allow in-depth investigations [5, 6]. For example, various public repositories [7–9] host protein, gene ontology and pathway information for chicken in addition to specialized chicken databases which are also freely accessible [10–12].
We recognized the need to greatly expand and integrate the information-base for Northern bobwhite to develop the species as a robust avian-wildlife genomic model. The Quail knowledgebase contains 478,142 raw ESTs, assembled nucleotide and protein sequences, and 21,980 unigenes annotated with meta-data. Data entities such as protein information and gene ontology annotation are integrated in a common platform with a web interface for comprehensive parameter searching. The linkage among these data entities is provided by unigene ID that connects the entities internally allowing the user to perform flexible query searches. The result of our effort is a web-accessible knowledgebase for Northern bobwhite which includes user friendly navigation tools and provides EST assembling information, sequence and structural properties and complex search utilities, bundled with an alternative method to generate sequence scaffolds to “stitch” transcripts against a reference organism. The data represented in the Quail Genomics knowledgebase have provided novel insights into the systemic perturbations of 2,6-DNT in Northern bobwhite . Similarly, the knowledgebase can provide researchers the ability to perform analysis and curate genomic information to further their own research pursuits.
Construction and content
Database schema and implementation
Nucleotide assembly and annotation
The preparation of nucleotide assembly data resulted in 71,384 unique sequences . All sequences were annotated against non-redundant protein database with BLASTX and also against model organism and chicken database using Parallel Blast  with HPC. Prediction of protein-coding regions was established using ESTScan which uses a hidden Markov model to identify coding regions that uses Gallus gallus as training data . The predicted protein-coding regions were scanned for protein signatures using Interproscan for high throughput annotation [19, 20]. The output is stored in the database and can be retrieved via querying for visual inspection of protein domains in HTML format. Hyperlinks are provided on the display results for retrieval of additional information. Microarray-based gene expression data is also included in the Quail Genomics knowledgebase where p-value and fold changes are stored as persistent data for each experiment. A full description of microarray experiments and analyses is provided in Rawat et al .
Gene Ontology browser
We performed reciprocal blast hit (RBH) to assess ortholog detection among Northern bobwhite and chicken (Gallus gallus, an intra-order phylogenetic relative of Northern bobwhite) across both the putative homologs (BLASTX, E ≤ 10-5) and predicted ORFs (ESTScan) that might lead to orthologous genes (Figure 2). We observed that putative homolog-ORF pairs (where matching protein identifiers were sorted on minimum E value for blast hits) were complementary in 85% of comparisons to the RBH pairs. To maximize the ortholog count for the Northern bobwhite, we conjoined ortholog sets derived from each method resulting in non-redundant orthologous unique transcripts that represent 8,825 unique gene products (Figure 2). To functionally annotate the 8,825 Northern bobwhite orthologs, we investigated and inferred putative GO  annotations finding matching annotations for 4,786 (54.2%) genes. We implemented the GO-Dev project from Amigo locally to provide Tree Browser to represent the Northern bobwhite orthologs.
GO-Dev is an open-source platform that consists of CGI/perl modules, database structure and web interface . GO-Dev and dependencies including GraphViz  which are required to successfully run the Amigo infrastructure locally were downloaded and installed. The Amigo infrastructure runs on the Quail Genomics webserver and interacts with our local MySQL database where database dumps are imported into the GO table structures. We exported our GO data for Northern bobwhite orthologs to the local Amigo database and the data can be viewed via tree browser from Quail Genomics.
The 71,384 unigenes identified for Northern bobwhite are an over representation of the total protein-coding genes that are expected to make up the Northern bobwhite genome. Frequently, due to missing EST sequences, the ESTs from a single gene may not overlap to assemble to a contiguous sequence resulting in non-overlapping contigs and singletons or splits in genes. Also, stringent parameters during assembly of ESTs into contigs might lead to unassembled sequences especially when the sequences have low genome coverage . These issues of missing sequences or fragmentation might lead to partial representation of a protein-coding sequence. Many of the sequence fragments might actually represent the same protein leading to redundancy in the assembled sequences.
For model organisms, methods such as Blat  and Bowtie  can be utilized to annotate against a reference genome; however inadequate representation of a reference sequence for a non-model organism makes annotation difficult. Other methods like Ensemble gene-build pipeline  are also available however these do not allow user to select ‘gene of interest’ and allow visualization. Finally, methods such as Genescript  do provide visualization features, however integration of these with our web interface and database is not practical due to workflow and operating system incompatibility.
It may be of interest to further study these scaffolds to understand redundancy or potential alternate spliced elements. In conjunction with Parallel Blast output stored as persistent data and the sequence information stored in relational database management system (RDBMS), our pipeline allows users to interact and visualize results ‘on the fly’. The temporary file handler added in the script allows multi-user access and deletes files created during scaffold building, to maintain housekeeping on the server.
Utility and discussion
Composition of data available in Quail Genomics knowledgebase.
Data Content in Quail Genomics
71,384 Unigenes (35,904 contigs, 35,480 singlets)
Predicted protein regions
39,400 potential ORFs
15,057 Interproscan hits
8,825 putative orthologs
4,786 GO terms
Beside annotation search, we have integrated additional searches in our platform. Users can search ESTs that assemble as contigs and visualize the overlap and direction against the assembly. Users can also input their sequence and perform blast search (BLASTN, TBLASTX) against the indexed nucleotide sequences of Northern bobwhite. The expression data is stored based on experiment and dose and output can be viewed for microarray probes sorted on p-value. The microarray probes which had a statistically significant increase or decrease in expression (p-value<.05) are highlighted in red or green, respectively.
The Northern bobwhite orthologs are functionally annotated under the Inferred from the electronic annotation (IEA) evidence level. The GO categories of these orthologs can be browsed for biological processes, cellular components, and molecular functions through the GO Tree Browser implemented from Amigo . With guidelines as defined by GO consortium , these orthologs are candidates that might be considered for update to Inferred from Sequence Orthology (ISO) evidence level.
Past applications and results
The data represented in Quail Genomics knowledgebase have provided insights into the metabolic perturbations underlying several observed toxicological phenotypes in a 2,6-DNT-exposure case study investigating Northern bobwhite . The comprehensive metadata attributes helped us to identify RT-qPCR validated impacts.
Japanese quail, a reproductive avian model species , has only 136 ESTs available in GenBank (May’2010). The Quail Genomics knowledgebase will soon incorporate 559,819 ESTs generated for Japanese quail by next generation sequencing using GS-FLX technology (454 Life Sciences / Roche, Branford, CT) developed to further ecotoxicological research in Japanese quail (unpublished). Subsequent updates will be performed every 3 months or as required. We will continue to work on the scaffold module allowing users to interact with output and download in GFF format.
The Quail Genomics knowledgebase provides a web-based utility for genomic investigations in an emerging wildlife model, the Northern bobwhite. As of March 2010, this knowledgebase contains raw sequence, assembled sequence, annotations and gene expression data for the Northern bobwhite. The Quail Genomics knowledgebase will be integrated with Japanese quail genomics and incorporated into a broader platform for investigations of avian species.
Availability and requirements
Quail Genomics knowledgebase is publicly available . The web interface is HTML 4.01 and has been tested with Firefox 3 and Internet Explorer 7. PERL, PHP, GO-DEV, MYSQL, BioPERL 1.6 are supported by dependencies and run on Quad core, 16GB RAM, MAC OSX 1.6. Raw data and microarray data have been deposited in public repositories and can be downloaded from the links provided.
We gratefully acknowledge the support of the Mississippi Functional Genomics Network (NIH/NCRR P20 RR016476) and grant #W912HZ-08-C-0032 from the U. S. Army Environmental Quality/Installations Research Program. We thank Glover George for assisting in establishing MPI-Blast and use of http://cluster.vislab.usm.edu. Permission was granted from the Chief of Engineers to publish this information.
This article has been published as part of BMC Bioinformatics Volume 11 Supplement 6, 2010: Proceedings of the Seventh Annual MCBIOS Conference. Bioinformatics: Systems, Biology, Informatics and Computation. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/11?issue=S6.
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