In recent years, the emphasis of scientific inquiry has shifted from whole-genome analyses to an understanding of cellular responses specific to tissue, developmental stage or environmental conditions. One of the central mechanisms underlying the diversity and adaptability of the contextual responses is alternative splicing (AS). It enables a single gene to encode multiple isoforms with distinct biological functions. However, to date, the functions of the vast majority of differentially spliced protein isoforms are not known. Integration of genomic, proteomic, functional, phenotypic and contextual information is essential for supporting isoform-based modeling and analysis. Such integrative proteogenomics approaches promise to provide insights into the functions of the alternatively spliced protein isoforms and provide high-confidence hypotheses to be validated experimentally. This manuscript provides a survey of the public databases supporting isoform-based biology. It also presents an overview of the potential global impact of AS on the human canonical gene functions, molecular interactions and cellular pathways.

Metagenomic sequencing has produced significant amounts of data in recent years. For example, as of summer 2013, MG-RAST has been used to annotate over 110,000 data sets totaling over 43 Terabases. With metagenomic sequencing finding even wider adoption in the scientific community, the existing web-based analysis tools and infrastructure in MG-RAST provide limited capability for data retrieval and analysis, such as comparative analysis between multiple data sets. Moreover, although the system provides many analysis tools, it is not comprehensive. By opening MG-RAST up via a web services API (application programmers interface) we have greatly expanded access to MG-RAST data, as well as provided a mechanism for the use of third-party analysis tools with MG-RAST data. This RESTful API makes all data and data objects created by the MG-RAST pipeline accessible as JSON objects. As part of the DOE Systems Biology Knowledgebase project (KBase, http://kbase.us) we have implemented a web services API for MG-RAST. This API complements the existing MG-RAST web interface and constitutes the basis of KBase's microbial community capabilities. In addition, the API exposes a comprehensive collection of data to programmers. This API, which uses a RESTful (Representational State Transfer) implementation, is compatible with most programming environments and should be easy to use for end users and third parties. It provides comprehensive access to sequence data, quality control results, annotations, and many other data types. Where feasible, we have used standards to expose data and metadata. Code examples are provided in a number of languages both to show the versatility of the API and to provide a starting point for users. We present an API that exposes the data in MG-RAST for consumption by our users, greatly enhancing the utility of the MG-RAST service.

BACKGROUND: Gene prediction algorithms (or gene callers) are an essential tool for analyzing shotgun nucleic acid sequence data. Gene prediction is a ubiquitous step in sequence analysis pipelines; it reduces the volume of data by identifying the most likely reading frame for a fragment, permitting the out-of-frame translations to be ignored. In this study we evaluate five widely used ab initio gene-calling algorithms-FragGeneScan, MetaGeneAnnotator, MetaGeneMark, Orphelia, and Prodigal-for accuracy on short (75-1000 bp) fragments containing sequence error from previously published artificial data and "real" metagenomic datasets. RESULTS: While gene prediction tools have similar accuracies predicting genes on error-free fragments, in the presence of sequencing errors considerable differences between tools become evident. For error-containing short reads, FragGeneScan finds more prokaryotic coding regions than does MetaGeneAnnotator, MetaGeneMark, Orphelia, or Prodigal. This improved detection of genes in error-containing fragments, however, comes at the cost of much lower (50%) specificity and overprediction of genes in noncoding regions. CONCLUSIONS: Ab initio gene callers offer a significant reduction in the computational burden of annotating individual nucleic acid reads and are used in many metagenomic annotation systems. For predicting reading frames on raw reads, we find the hidden Markov model approach in FragGeneScan is more sensitive than other gene prediction tools, while Prodigal, MGA, and MGM are better suited for higher-quality sequences such as assembled contigs.

We provide a novel method, DRISEE (duplicate read inferred sequencing error estimation), to assess sequencing quality (alternatively referred to as "noise" or "error") within and/or between sequencing samples. DRISEE provides positional error estimates that can be used to inform read trimming within a sample. It also provides global (whole sample) error estimates that can be used to identify samples with high or varying levels of sequencing error that may confound downstream analyses, particularly in the case of studies that utilize data from multiple sequencing samples. For shotgun metagenomic data, we believe that DRISEE provides estimates of sequencing error that are more accurate and less constrained by technical limitations than existing methods that rely on reference genomes or the use of scores (e.g. Phred). Here, DRISEE is applied to (non amplicon) data sets from both the 454 and Illumina platforms. The DRISEE error estimate is obtained by analyzing sets of artifactual duplicate reads (ADRs), a known by-product of both sequencing platforms. We present DRISEE as an open-source, platform-independent method to assess sequencing error in shotgun metagenomic data, and utilize it to discover previously uncharacterized error in de novo sequence data from the 454 and Illumina sequencing platforms.

BACKGROUND: Random community genomes (metagenomes) are now commonly used to study microbes in different environments. Over the past few years, the major challenge associated with metagenomics shifted from generating to analyzing sequences. High-throughput, low-cost next-generation sequencing has provided access to metagenomics to a wide range of researchers. RESULTS: A high-throughput pipeline has been constructed to provide high-performance computing to all researchers interested in using metagenomics. The pipeline produces automated functional assignments of sequences in the metagenome by comparing both protein and nucleotide databases. Phylogenetic and functional summaries of the metagenomes are generated, and tools for comparative metagenomics are incorporated into the standard views. User access is controlled to ensure data privacy, but the collaborative environment underpinning the service provides a framework for sharing datasets between multiple users. In the metagenomics RAST, all users retain full control of their data, and everything is available for download in a variety of formats. CONCLUSION: The open-source metagenomics RAST service provides a new paradigm for the annotation and analysis of metagenomes. With built-in support for multiple data sources and a back end that houses abstract data types, the metagenomics RAST is stable, extensible, and freely available to all researchers. This service has removed one of the primary bottlenecks in metagenome sequence analysis - the availability of high-performance computing for annotating the data. http://metagenomics.nmpdr.org.

Recent progress in genomics and experimental biology has brought exponential growth of the biological information available for computational analysis in public genomics databases. However, applying the potentially enormous scientific value of this information to the understanding of biological systems requires computing and data storage technology of an unprecedented scale. The grid, with its aggregated and distributed computational and storage infrastructure, offers an ideal platform for high-throughput bioinformatics analysis. To leverage this we have developed the Genome Analysis Research Environment (GNARE) - a scalable computational system for the high-throughput analysis of genomes, which provides an integrated database and computational backend for data-driven bioinformatics applications. GNARE efficiently automates the major steps of genome analysis including acquisition of data from multiple genomic databases; data analysis by a diverse set of bioinformatics tools; and storage of results and annotations. High-throughput computations in GNARE are performed using distributed heterogeneous grid computing resources such as Grid2003, TeraGrid, and the DOE science grid. Multi-step genome analysis workflows involving massive data processing, the use of application-specific toots and algorithms and updating of an integrated database to provide interactive Web access to results are all expressed and controlled by a "virtual data" model which transparently maps computational workflows to distributed grid resources. This paper describes how Grid technologies such as Globus, Condor, and the Gryphyn virtual data system were applied in the development of GNARE. It focuses on our approach to Grid resource allocation and to the use of GNARE as a computational framework for the development of bioinformatics applications.

MOTIVATION: The identification of sequence patterns involved in gene regulation and expression is a major challenge in molecular biology. In this paper we describe a novel algorithm and the software for searching nucleotide and protein sequences for complex nucleotide patterns including potential secondary structure elements, also allowing for mismatches/mispairings below a user-fixed threshold, and assessing the statistical significance of their occurrence through a Markov chain simulation. RESULTS: The application of the proposed algorithm allowed the identification of some functional elements, such as the Iron Responsive Element, the Histone stem-loop structure and the Selenocysteine Insertion Sequence, located in the mRNA untranslated regions of post-transcriptionally regulated genes with the assessment of sensitivity and selectivity of the searching method. AVAILABILITY: A Web interface is available at: http://bigarea.area.ba.cnr.it:8000/EmbIT/Pats earch.html.