Veronika Vonstein

The RAST Server: Rapid Annotations using Subsystems Technology

BackgroundThe number of prokaryotic genome sequences becoming available is growing steadily and is growing faster than our ability to accurately annotate them.DescriptionWe describe a fully automated service for annotating bacterial and archaeal genomes. The service identifies protein-encoding, rRNA and tRNA genes, assigns functions to the genes, predicts which subsystems are represented in the genome, uses this information to reconstruct the metabolic network and makes the output easily downloadable for the user. In addition, the annotated genome can be browsed in an environment that supports comparative analysis with the annotated genomes maintained in the SEED environment.The service normally makes the annotated genome available within 12–24 hours of submission, but ultimately the quality of such a service will be judged in terms of accuracy, consistency, and completeness of the produced annotations. We summarize our attempts to address these issues and discuss plans for incrementally enhancing the service.ConclusionBy providing accurate, rapid annotation freely to the community we have created an important community resource. The service has now been utilized by over 120 external users annotating over 350 distinct genomes.

The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST)

In 2004, the SEED (http://pubseed.theseed.org/) was created to provide consistent and accurate genome annotations across thousands of genomes and as a platform for discovering and developing de novo annotations. The SEED is a constantly updated integration of genomic data with a genome database, web front end, API and server scripts. It is used by many scientists for predicting gene functions and discovering new pathways. In addition to being a powerful database for bioinformatics research, the SEED also houses subsystems (collections of functionally related protein families) and their derived FIGfams (protein families), which represent the core of the RAST annotation engine (http://rast.nmpdr.org/). When a new genome is submitted to RAST, genes are called and their annotations are made by comparison to the FIGfam collection. If the genome is made public, it is then housed within the SEED and its proteins populate the FIGfam collection. This annotation cycle has proven to be a robust and scalable solution to the problem of annotating the exponentially increasing number of genomes. To date, >12 000 users worldwide have annotated >60 000 distinct genomes using RAST. Here we describe the interconnectedness of the SEED database and RAST, the RAST annotation pipeline and updates to both resources.

The Subsystems Approach to Genome Annotation and its Use in the Project to Annotate 1000 Genomes

The release of the 1000th complete microbial genome will occur in the next two to three years. In anticipation of this milestone, the Fellowship for Interpretation of Genomes (FIG) launched the Project to Annotate 1000 Genomes. The project is built around the principle that the key to improved accuracy in high-throughput annotation technology is to have experts annotate single subsystems over the complete collection of genomes, rather than having an annotation expert attempt to annotate all of the genes in a single genome. Using the subsystems approach, all of the genes implementing the subsystem are analyzed by an expert in that subsystem. An annotation environment was created where populated subsystems are curated and projected to new genomes. A portable notion of a populated subsystem was defined, and tools developed for exchanging and curating these objects. Tools were also developed to resolve conflicts between populated subsystems. The SEED is the first annotation environment that supports this model of annotation. Here, we describe the subsystem approach, and offer the first release of our growing library of populated subsystems. The initial release of data includes 180 177 distinct proteins with 2133 distinct functional roles. This data comes from 173 subsystems and 383 different organisms.

RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes

The RAST (Rapid Annotation using Subsystem Technology) annotation engine was built in 2008 to annotate bacterial and archaeal genomes. It works by offering a standard software pipeline for identifying genomic features (i.e., protein-encoding genes and RNA) and annotating their functions. Recently, in order to make RAST a more useful research tool and to keep pace with advancements in bioinformatics, it has become desirable to build a version of RAST that is both customizable and extensible. In this paper, we describe the RAST tool kit (RASTtk), a modular version of RAST that enables researchers to build custom annotation pipelines. RASTtk offers a choice of software for identifying and annotating genomic features as well as the ability to add custom features to an annotation job. RASTtk also accommodates the batch submission of genomes and the ability to customize annotation protocols for batch submissions. This is the first major software restructuring of RAST since its inception.

The ERGOTM genome analysis and discovery system

The ERGO (http://ergo.integratedgenomics.com/ERGO/) genome analysis and discovery suite is an integration of biological data from genomics, biochemistry, high-throughput expression profiling, genetics and peer-reviewed journals to achieve a comprehensive analysis of genes and genomes. Far beyond any conventional systems that facilitate functional assignments, ERGO combines pattern-based analysis with comparative genomics by visualizing genes within the context of regulation, expression profiling, phylogenetic clusters, fusion events, networked cellular pathways and chromosomal neighborhoods of other functionally related genes. The result of this multifaceted approach is to provide an extensively curated database of the largest available integration of genomes, with a vast collection of reconstructed cellular pathways spanning all domains of life. Although access to ERGO is provided only under subscription, it is already widely used by the academic community. The current version of the system integrates 500 genomes from all domains of life in various levels of completion, 403 of which are available for subscription.

Characterization of the Staphylococcus aureus Heat Shock, Cold Shock, Stringent, and SOS Responses and Their Effects on Log-Phase mRNA Turnover

Despite its being a leading cause of nosocomal and community-acquired infections, surprisingly little is known about Staphylococcus aureus stress responses. In the current study, Affymetrix S. aureus GeneChips were used to define transcriptome changes in response to cold shock, heat shock, stringent, and SOS response-inducing conditions. Additionally, the RNA turnover properties of each response were measured. Each stress response induced distinct biological processes, subsets of virulence factors, and antibiotic determinants. The results were validated by real-time PCR and stress-mediated changes in antimicrobial agent susceptibility. Collectively, many S. aureus stress-responsive functions are conserved across bacteria, whereas others are unique to the organism. Sets of small stable RNA molecules with no open reading frames were also components of each response. Induction of the stringent, cold shock, and heat shock responses dramatically stabilized most mRNA species. Correlations between mRNA turnover properties and transcript titers suggest that S. aureus stress response-dependent alterations in transcript abundances can, in part, be attributed to alterations in RNA stability. This phenomenon was not observed within SOS-responsive cells.

Improvements to PATRIC, the all-bacterial Bioinformatics Database and Analysis Resource Center

The Pathosystems Resource Integration Center (PATRIC) is the bacterial Bioinformatics Resource Center (https://www.patricbrc.org). Recent changes to PATRIC include a redesign of the web interface and some new services that provide users with a platform that takes them from raw reads to an integrated analysis experience. The redesigned interface allows researchers direct access to tools and data, and the emphasis has changed to user-created genome-groups, with detailed summaries and views of the data that researchers have selected. Perhaps the biggest change has been the enhanced capability for researchers to analyze their private data and compare it to the available public data. Researchers can assemble their raw sequence reads and annotate the contigs using RASTtk. PATRIC also provides services for RNA-Seq, variation, model reconstruction and differential expression analysis, all delivered through an updated private workspace. Private data can be compared by ‘virtual integration’ to any of PATRICs public data. The number of genomes available for comparison in PATRIC has expanded to over 80 000, with a special emphasis on genomes with antimicrobial resistance data. PATRIC uses this data to improve both subsystem annotation and k-mer classification, and tags new genomes as having signatures that indicate susceptibility or resistance to specific antibiotics.

The National Microbial Pathogen Database Resource (NMPDR): a genomics platform based on subsystem annotation

The National Microbial Pathogen Data Resource (NMPDR) () is a National Institute of Allergy and Infections Disease (NIAID)-funded Bioinformatics Resource Center that supports research in selected Category B pathogens. NMPDR contains the complete genomes of ∼50 strains of pathogenic bacteria that are the focus of our curators, as well as >400 other genomes that provide a broad context for comparative analysis across the three phylogenetic Domains. NMPDR integrates complete, public genomes with expertly curated biological subsystems to provide the most consistent genome annotations. Subsystems are sets of functional roles related by a biologically meaningful organizing principle, which are built over large collections of genomes; they provide researchers with consistent functional assignments in a biologically structured context. Investigators can browse subsystems and reactions to develop accurate reconstructions of the metabolic networks of any sequenced organism. NMPDR provides a comprehensive bioinformatics platform, with tools and viewers for genome analysis. Results of precomputed gene clustering analyses can be retrieved in tabular or graphic format with one-click tools. NMPDR tools include Signature Genes, which finds the set of genes in common or that differentiates two groups of organisms. Essentiality data collated from genome-wide studies have been curated. Drug target identification and high-throughput, in silico, compound screening are in development.

Accessing the SEED Genome Databases via Web Services API: Tools for Programmers

BackgroundThe SEED integrates many publicly available genome sequences into a single resource. The database contains accurate and up-to-date annotations based on the subsystems concept that leverages clustering between genomes and other clues to accurately and efficiently annotate microbial genomes. The backend is used as the foundation for many genome annotation tools, such as the Rapid Annotation using Subsystems Technology (RAST) server for whole genome annotation, the metagenomics RAST server for random community genome annotations, and the annotation clearinghouse for exchanging annotations from different resources. In addition to a web user interface, the SEED also provides Web services based API for programmatic access to the data in the SEED, allowing the development of third-party tools and mash-ups.ResultsThe currently exposed Web services encompass over forty different methods for accessing data related to microbial genome annotations. The Web services provide comprehensive access to the database back end, allowing any programmer access to the most consistent and accurate genome annotations available. The Web services are deployed using a platform independent service-oriented approach that allows the user to choose the most suitable programming platform for their application. Example code demonstrate that Web services can be used to access the SEED using common bioinformatics programming languages such as Perl, Python, and Java.ConclusionsWe present a novel approach to access the SEED database. Using Web services, a robust API for access to genomics data is provided, without requiring large volume downloads all at once. The API ensures timely access to the most current datasets available, including the new genomes as soon as they come online.

SEED Servers: High-Performance Access to the SEED Genomes, Annotations, and Metabolic Models

The remarkable advance in sequencing technology and the rising interest in medical and environmental microbiology, biotechnology, and synthetic biology resulted in a deluge of published microbial genomes. Yet, genome annotation, comparison, and modeling remain a major bottleneck to the translation of sequence information into biological knowledge, hence computational analysis tools are continuously being developed for rapid genome annotation and interpretation. Among the earliest, most comprehensive resources for prokaryotic genome analysis, the SEED project, initiated in 2003 as an integration of genomic data and analysis tools, now contains >5,000 complete genomes, a constantly updated set of curated annotations embodied in a large and growing collection of encoded subsystems, a derived set of protein families, and hundreds of genome-scale metabolic models. Until recently, however, maintaining current copies of the SEED code and data at remote locations has been a pressing issue. To allow high-performance remote access to the SEED database, we developed the SEED Servers (http://www.theseed.org/servers): four network-based servers intended to expose the data in the underlying relational database, support basic annotation services, offer programmatic access to the capabilities of the RAST annotation server, and provide access to a growing collection of metabolic models that support flux balance analysis. The SEED servers offer open access to regularly updated data, the ability to annotate prokaryotic genomes, the ability to create metabolic reconstructions and detailed models of metabolism, and access to hundreds of existing metabolic models. This work offers and supports a framework upon which other groups can build independent research efforts. Large integrations of genomic data represent one of the major intellectual resources driving research in biology, and programmatic access to the SEED data will provide significant utility to a broad collection of potential users.