DNA sequencing technology using chain-terminating dideoxy nucleoside triphosphates, first developed by Frederick Sanger [1
], has remained the mainstay of genome sequencing efforts for more than thirty years. However, recently developed, new massively parallel DNA sequencing platforms are now extensively used to generate sequence data at a fraction of the cost and labor required by Sanger technology. Three "next generation" sequencing systems that are currently commercially available include the Roche/454 Genome Sequencer [3
], Illumina/Solexa Genome Analyzer II [4
] and Applied Biosystems SOLiD System [6
]. In addition, commercial release of two additional platforms, including the Helicos Heliscope and the Pacific Biosmart SMRT, are planned for 2010 [7
Collectively, these systems, with their high depth of coverage and relatively low costs, have allowed individual researchers to initiate genome sequencing projects that were previously available to only large genome centers [8
]. The enhanced sequencing capability afforded by next-generation sequencing has had an especially significant impact on bacterial genomics. By facilitating genome sequencing of multiple isolates of the same bacterial species, several examples of extensive intraspecies genotypic heterogeneity have been revealed, leading to a revision of many long-standing views of microbial speciation [11
]. One of the first such studies revealed significant genetic variability among eight different strains of Streptococcus agalactiae
, group B Streptococcus
]. After performing cross strain comparisons Tettelin et al
. found a considerable number of genes not shared among the strains. Their discovery led to the proposal of the bacterial "pan-genome", defined as the global gene repertoire of a bacterial species comprised of the core genome (the set of genes shared by all the strains of the same bacterial species), the dispensable genome (the set of genes present in some but not all of the strains) and the strain specific genes (the set of genes found only in a single strain) [14
]. Genome heterogeneity has also been noted for species of Helicobacter pylori
, Staphylococcus aureus
, and Escherichia coli
]. As noted by Muzzi et al
., comparative genomics of bacterial species has important implications for vaccine development and discovery of novel antimicrobials [17
]. Other novel applications for next generation sequencing technologies have also been developed, including bacterial metagenomics [18
], and transcriptome mapping [21
Despite the potential for new insights into bacterial diversity and function, important challenges continue to include the organization, management and analysis of genome sequencing data. To address the need for tools for querying, analyzing and comparing multiple genomes of related species, several databases and software tools have been developed [25
], including the Integrated Microbial Genomes (IMG) system [26
], Integrated Microbial Genomes-Expert Review (IMG ER) system [28
], GenColors [29
], the Microbial Genome Database (MBGD) [31
], the Comprehensive Microbial Resource (CMR) [33
] and the EDGAR software [34
The IMG system contains complete and draft microbial genome sequence data generated by the Joint Genomes Institute (JGI) as well as other publicly available genome data not limited to microorganisms. Tools provided through IMG allow users to query, view and perform comparative analysis of genomes, genes and functions. Recently, a new version of IMG called IMG ER has been added to the IMG system. Tools available through IMG ER allow users to analyze and curate annotated microbial genome data whether it is unpublished or published. Although IMG ER allows users to upload their genome sequencing data for curation and analysis, it is not available for download and in-house use. The GenColors software allows users to browse, analyze and compare genome information from complete and ongoing genome projects related to prokaryotic or eukaryotic genomes. Additionally, GenColors may be used for the purpose of annotation in the case of incomplete projects. The CMR software contains sequence and annotation data for all of the current publicly available completed microbial genomes and provides a variety of comparison tools for the analysis of the multiple genomes including cross-genome analysis capabilities. Currently, however, there is no functionality that allows users to submit genome data for use with CMR. Similar to CMR, MBGD provides users with several tools for the comparison and analysis of complete bacterial genomes. Unlike CMR, MBGD contains a newly added feature called MyMBGD that allows users to add their own genome data to MBGD. The EDGAR software has recently been released and includes comparative analysis tools for the comparison of multiple strains of a given species. EDGAR offers similar capabilities to those found in CMR and MBGD, in addition to features such as phylogenetic analyses and visualization capabilities including Venn diagrams and synteny plots.
While the aforementioned systems include data management and analysis functionalities there are limitations. For example, genome projects that include proprietary data may be restricted in the submission of the data to third party software. Many of the current data management software tools are not available for download and in-house use, a requirement when access to next generation sequencing instruments can outstrip the availability of experienced bioinformaticians to assist with data management and analysis.
In addition to the already mentioned software applications, there are other tools that are designed for genome annotation or re-annotation of unpublished or published genomes [25
]. Several of these tools provide data curation capabilities for the purpose of correcting annotation errors and improving annotated data but are restricted to use with the annotated data generated through specified software packages. Additionally, as with many software applications, they require the researcher to develop a working knowledge of the analysis capabilities of the software as well as provide "expert" curation of the data. With the increased use of next-generation sequencing in academic, industrial and government settings, however, biologists do not always have immediate access to computational support needed to easily manage the data and to initiate comparative analysis.
To overcome some of these limitations, DraGnET was developed specifically to provide biologists with their own web based tool that is both convenient and easy to use. DraGnET allows researchers to independently store, retrieve and curate their own data generated from any annotation engine and to perform genome comparisons during the beginning phase of a sequencing project. Additionally, publicly available genome data can be stored for the purpose of comparing draft genome data with reference genomes. DraGnET includes provisions for data access, searching, and modification as well as access to basic local alignment search tool (BLAST) functionalities [37
] for amino acid sequence similarity searches and cross strain comparisons. As a consequence, DraGnET allows investigators to immediately begin testing of biologically relevant hypotheses without having to devote time to learning sophisticated analysis programs or to depend on computational support from designated personnel. Additionally, the DraGnET source code has been made available, allowing researchers to further customize and develop the software to meet the needs of specific sequencing projects.
To demonstrate the utility of DraGnET, we have successfully established a DraGnET project, deployed for Internet access, and performed preliminary cross strain comparisons to identify potential vaccine targets against the animal pathogen Haemophilus parasuis
. Microbial genome sequencing has proven to be a powerful approach to identify new, protective vaccines via reverse vaccinology
, i.e., discovery of vaccine targets by scanning sequence data for potential surface-exposed antigens [38
]. Moreover, broadly protective antigens may be identified by comparison of genomes from multiple strains of a single species [17
]. Reverse vaccinology has led to the development of new vaccines for several human and animal pathogens where previously vaccines were not available [41
]. DraGnET enables facile preliminary comparisons of multiple draft or complete genome sequences of any number of organisms, including identification of protein encoding genes shared by multiple strains, making DraGnET a useful bioinformatic tool.