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1.  An Integrated Approach for Analyzing Clinical Genomic Variant Data from Next-Generation Sequencing 
Next-generation sequencing (NGS) technologies provide the potential for developing high-throughput and low-cost platforms for clinical diagnostics. A limiting factor to clinical applications of genomic NGS is downstream bioinformatics analysis for data interpretation. We have developed an integrated approach for end-to-end clinical NGS data analysis from variant detection to functional profiling. Robust bioinformatics pipelines were implemented for genome alignment, single nucleotide polymorphism (SNP), small insertion/deletion (InDel), and copy number variation (CNV) detection of whole exome sequencing (WES) data from the Illumina platform. Quality-control metrics were analyzed at each step of the pipeline by use of a validated training dataset to ensure data integrity for clinical applications. We annotate the variants with data regarding the disease population and variant impact. Custom algorithms were developed to filter variants based on criteria, such as quality of variant, inheritance pattern, and impact of variant on protein function. The developed clinical variant pipeline links the identified rare variants to Integrated Genome Viewer for visualization in a genomic context and to the Protein Information Resource’s iProXpress for rich protein and disease information. With the application of our system of annotations, prioritizations, inheritance filters, and functional profiling and analysis, we have created a unique methodology for downstream variant filtering that empowers clinicians and researchers to interpret more effectively the relevance of genomic alterations within a rare genetic disease.
doi:10.7171/jbt.15-2601-002
PMCID: PMC4310222  PMID: 25649353
bioinformatics; genetic alterations; Mendelian Genetics; protein information resources
2.  Elevated FGF21 secretion, PGC-1α and ketogenic enzyme expression are hallmarks of iron–sulfur cluster depletion in human skeletal muscle 
Human Molecular Genetics  2013;23(1):24-39.
Iron–sulfur (Fe-S) clusters are ancient enzyme cofactors found in virtually all life forms. We evaluated the physiological effects of chronic Fe-S cluster deficiency in human skeletal muscle, a tissue that relies heavily on Fe-S cluster-mediated aerobic energy metabolism. Despite greatly decreased oxidative capacity, muscle tissue from patients deficient in the Fe-S cluster scaffold protein ISCU showed a predominance of type I oxidative muscle fibers and higher capillary density, enhanced expression of transcriptional co-activator PGC-1α and increased mitochondrial fatty acid oxidation genes. These Fe-S cluster-deficient muscles showed a dramatic up-regulation of the ketogenic enzyme HMGCS2 and the secreted protein FGF21 (fibroblast growth factor 21). Enhanced muscle FGF21 expression was reflected by elevated circulating FGF21 levels in the patients, and robust FGF21 secretion could be recapitulated by respiratory chain inhibition in cultured myotubes. Our findings reveal that mitochondrial energy starvation elicits a coordinated response in Fe-S-deficient skeletal muscle that is reflected systemically by increased plasma FGF21 levels.
doi:10.1093/hmg/ddt393
PMCID: PMC3857942  PMID: 23943793
3.  A fast Peptide Match service for UniProt Knowledgebase 
Bioinformatics  2013;29(21):2808-2809.
Summary: We have developed a new web application for peptide matching using Apache Lucene-based search engine. The Peptide Match service is designed to quickly retrieve all occurrences of a given query peptide from UniProt Knowledgebase (UniProtKB) with isoforms. The matched proteins are shown in summary tables with rich annotations, including matched sequence region(s) and links to corresponding proteins in a number of proteomic/peptide spectral databases. The results are grouped by taxonomy and can be browsed by organism, taxonomic group or taxonomy tree. The service supports queries where isobaric leucine and isoleucine are treated equivalent, and an option for searching UniRef100 representative sequences, as well as dynamic queries to major proteomic databases. In addition to the web interface, we also provide RESTful web services. The underlying data are updated every 4 weeks in accordance with the UniProt releases.
Availability: http://proteininformationresource.org/peptide.shtml
Contact: chenc@udel.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt484
PMCID: PMC3799477  PMID: 23958731
4.  RLIMS-P: an online text-mining tool for literature-based extraction of protein phosphorylation information 
Protein phosphorylation is central to the regulation of most aspects of cell function. Given its importance, it has been the subject of active research as well as the focus of curation in several biological databases. We have developed Rule-based Literature Mining System for protein Phosphorylation (RLIMS-P), an online text-mining tool to help curators identify biomedical research articles relevant to protein phosphorylation. The tool presents information on protein kinases, substrates and phosphorylation sites automatically extracted from the biomedical literature. The utility of the RLIMS-P Web site has been evaluated by curators from Phospho.ELM, PhosphoGRID/BioGrid and Protein Ontology as part of the BioCreative IV user interactive task (IAT). The system achieved F-scores of 0.76, 0.88 and 0.92 for the extraction of kinase, substrate and phosphorylation sites, respectively, and a precision of 0.88 in the retrieval of relevant phosphorylation literature. The system also received highly favorable feedback from the curators in a user survey. Based on the curators’ suggestions, the Web site has been enhanced to improve its usability. In the RLIMS-P Web site, phosphorylation information can be retrieved by PubMed IDs or keywords, with an option for selecting targeted species. The result page displays a sortable table with phosphorylation information. The text evidence page displays the abstract with color-coded entity mentions and includes links to UniProtKB entries via normalization, i.e. the linking of entity mentions to database identifiers, facilitated by the GenNorm tool and by the links to the bibliography in UniProt. Log in and editing capabilities are offered to any user interested in contributing to the validation of RLIMS-P results. Retrieved phosphorylation information can also be downloaded in CSV format and the text evidence in the BioC format. RLIMS-P is freely available.
Database URL: http://www.proteininformationresource.org/rlimsp/
doi:10.1093/database/bau081
PMCID: PMC4131691  PMID: 25122463
5.  Software for pre-processing Illumina next-generation sequencing short read sequences 
Background
When compared to Sanger sequencing technology, next-generation sequencing (NGS) technologies are hindered by shorter sequence read length, higher base-call error rate, non-uniform coverage, and platform-specific sequencing artifacts. These characteristics lower the quality of their downstream analyses, e.g. de novo and reference-based assembly, by introducing sequencing artifacts and errors that may contribute to incorrect interpretation of data. Although many tools have been developed for quality control and pre-processing of NGS data, none of them provide flexible and comprehensive trimming options in conjunction with parallel processing to expedite pre-processing of large NGS datasets.
Methods
We developed ngsShoRT (next-generation sequencing Short Reads Trimmer), a flexible and comprehensive open-source software package written in Perl that provides a set of algorithms commonly used for pre-processing NGS short read sequences. We compared the features and performance of ngsShoRT with existing tools: CutAdapt, NGS QC Toolkit and Trimmomatic. We also compared the effects of using pre-processed short read sequences generated by different algorithms on de novo and reference-based assembly for three different genomes: Caenorhabditis elegans, Saccharomyces cerevisiae S288c, and Escherichia coli O157 H7.
Results
Several combinations of ngsShoRT algorithms were tested on publicly available Illumina GA II, HiSeq 2000, and MiSeq eukaryotic and bacteria genomic short read sequences with the focus on removing sequencing artifacts and low-quality reads and/or bases. Our results show that across three organisms and three sequencing platforms, trimming improved the mean quality scores of trimmed sequences. Using trimmed sequences for de novo and reference-based assembly improved assembly quality as well as assembler performance. In general, ngsShoRT outperformed comparable trimming tools in terms of trimming speed and improvement of de novo and reference-based assembly as measured by assembly contiguity and correctness.
Conclusions
Trimming of short read sequences can improve the quality of de novo and reference-based assembly and assembler performance. The parallel processing capability of ngsShoRT reduces trimming time and improves the memory efficiency when dealing with large datasets. We recommend combining sequencing artifacts removal, and quality score based read filtering and base trimming as the most consistent method for improving sequence quality and downstream assemblies.
ngsShoRT source code, user guide and tutorial are available at http://research.bioinformatics.udel.edu/genomics/ngsShoRT/. ngsShoRT can be incorporated as a pre-processing step in genome and transcriptome assembly projects.
doi:10.1186/1751-0473-9-8
PMCID: PMC4064128  PMID: 24955109
Next-generation sequencing; Illumina; Trimming; De novo assembly; Reference-based assembly; Perl
6.  Protein Ontology: a controlled structured network of protein entities 
Nucleic Acids Research  2013;42(Database issue):D415-D421.
The Protein Ontology (PRO; http://proconsortium.org) formally defines protein entities and explicitly represents their major forms and interrelations. Protein entities represented in PRO corresponding to single amino acid chains are categorized by level of specificity into family, gene, sequence and modification metaclasses, and there is a separate metaclass for protein complexes. All metaclasses also have organism-specific derivatives. PRO complements established sequence databases such as UniProtKB, and interoperates with other biomedical and biological ontologies such as the Gene Ontology (GO). PRO relates to UniProtKB in that PRO’s organism-specific classes of proteins encoded by a specific gene correspond to entities documented in UniProtKB entries. PRO relates to the GO in that PRO’s representations of organism-specific protein complexes are subclasses of the organism-agnostic protein complex terms in the GO Cellular Component Ontology. The past few years have seen growth and changes to the PRO, as well as new points of access to the data and new applications of PRO in immunology and proteomics. Here we describe some of these developments.
doi:10.1093/nar/gkt1173
PMCID: PMC3964965  PMID: 24270789
7.  Transcription factors and genetic circuits orchestrating the complex, multilayered response of Clostridium acetobutylicum to butanol and butyrate stress 
BMC Systems Biology  2013;7:120.
Background
Organisms of the genus Clostridium are Gram-positive endospore formers of great importance to the carbon cycle, human normo- and pathophysiology, but also in biofuel and biorefinery applications. Exposure of Clostridium organisms to chemical and in particular toxic metabolite stress is ubiquitous in both natural (such as in the human microbiome) and engineered environments, engaging both the general stress response as well as specialized programs. Yet, despite its fundamental and applied significance, it remains largely unexplored at the systems level.
Results
We generated a total of 96 individual sets of microarray data examining the transcriptional changes in C. acetobutylicum, a model Clostridium organism, in response to three levels of chemical stress from the native metabolites, butanol and butyrate. We identified 164 significantly differentially expressed transcriptional regulators and detailed the cellular programs associated with general and stressor-specific responses, many previously unexplored. Pattern-based, comparative genomic analyses enabled us, for the first time, to construct a detailed picture of the genetic circuitry underlying the stress response. Notably, a list of the regulons and DNA binding motifs of the stress-related transcription factors were identified: two heat-shock response regulators, HrcA and CtsR; the SOS response regulator LexA; the redox sensor Rex; and the peroxide sensor PerR. Moreover, several transcriptional regulators controlling stress-responsive amino acid and purine metabolism and their regulons were also identified, including ArgR (arginine biosynthesis and catabolism regulator), HisR (histidine biosynthesis regulator), CymR (cysteine metabolism repressor) and PurR (purine metabolism repressor).
Conclusions
Using an exceptionally large set of temporal transcriptional data and regulon analyses, we successfully built a STRING-based stress response network model integrating important players for the general and specialized metabolite stress response in C. acetobutylicum. Since the majority of the transcription factors and their target genes are highly conserved in other organisms of the Clostridium genus, this network would be largely applicable to other Clostridium organisms. The network informs the molecular basis of Clostridium responses to toxic metabolites in natural ecosystems and the microbiome, and will facilitate the construction of genome-scale models with added regulatory-network dimensions to guide the development of tolerant strains.
doi:10.1186/1752-0509-7-120
PMCID: PMC3828012  PMID: 24196194
Gene expression; Protein-protein interaction; Transcriptional regulatory network (TRN); Transcription factor (TF); TF binding site (TFBS); Transcriptional regulator (TR)
8.  A framework for biomedical figure segmentation towards image-based document retrieval 
BMC Systems Biology  2013;7(Suppl 4):S8.
The figures included in many of the biomedical publications play an important role in understanding the biological experiments and facts described within. Recent studies have shown that it is possible to integrate the information that is extracted from figures in classical document classification and retrieval tasks in order to improve their accuracy. One important observation about the figures included in biomedical publications is that they are often composed of multiple subfigures or panels, each describing different methodologies or results. The use of these multimodal figures is a common practice in bioscience, as experimental results are graphically validated via multiple methodologies or procedures. Thus, for a better use of multimodal figures in document classification or retrieval tasks, as well as for providing the evidence source for derived assertions, it is important to automatically segment multimodal figures into subfigures and panels. This is a challenging task, however, as different panels can contain similar objects (i.e., barcharts and linecharts) with multiple layouts. Also, certain types of biomedical figures are text-heavy (e.g., DNA sequences and protein sequences images) and they differ from traditional images. As a result, classical image segmentation techniques based on low-level image features, such as edges or color, are not directly applicable to robustly partition multimodal figures into single modal panels.
In this paper, we describe a robust solution for automatically identifying and segmenting unimodal panels from a multimodal figure. Our framework starts by robustly harvesting figure-caption pairs from biomedical articles. We base our approach on the observation that the document layout can be used to identify encoded figures and figure boundaries within PDF files. Taking into consideration the document layout allows us to correctly extract figures from the PDF document and associate their corresponding caption. We combine pixel-level representations of the extracted images with information gathered from their corresponding captions to estimate the number of panels in the figure. Thus, our approach simultaneously identifies the number of panels and the layout of figures.
In order to evaluate the approach described here, we applied our system on documents containing protein-protein interactions (PPIs) and compared the results against a gold standard that was annotated by biologists. Experimental results showed that our automatic figure segmentation approach surpasses pure caption-based and image-based approaches, achieving a 96.64% accuracy. To allow for efficient retrieval of information, as well as to provide the basis for integration into document classification and retrieval systems among other, we further developed a web-based interface that lets users easily retrieve panels containing the terms specified in the user queries.
doi:10.1186/1752-0509-7-S4-S8
PMCID: PMC3856606  PMID: 24565394
9.  Omics-Based Molecular Target and Biomarker Identification 
Genomic, proteomic, and other omic-based approaches are now broadly used in biomedical research to facilitate the understanding of disease mechanisms and identification of molecular targets and biomarkers for therapeutic and diagnostic development. While the Omics technologies and bioinformatics tools for analyzing Omics data are rapidly advancing, the functional analysis and interpretation of the data remain challenging due to the inherent nature of the generally long workflows of Omics experiments. We adopt a strategy that emphasizes the use of curated knowledge resources coupled with expert-guided examination and interpretation of Omics data for the selection of potential molecular targets. We describe a downstream workflow and procedures for functional analysis that focus on biological pathways, from which molecular targets can be derived and proposed for experimental validation.
doi:10.1007/978-1-61779-027-0_26
PMCID: PMC3742302  PMID: 21370102
Proteomics; Genomics; Bioinformatics; Biological pathways; Cell signaling; Databases; Molecular targets; Biomarkers
10.  Construction of protein phosphorylation networks by data mining, text mining and ontology integration: analysis of the spindle checkpoint 
Knowledge representation of the role of phosphorylation is essential for the meaningful understanding of many biological processes. However, such a representation is challenging because proteins can exist in numerous phosphorylated forms with each one having its own characteristic protein–protein interactions (PPIs), functions and subcellular localization. In this article, we evaluate the current state of phosphorylation event curation and then present a bioinformatics framework for the annotation and representation of phosphorylated proteins and construction of phosphorylation networks that addresses some of the gaps in current curation efforts. The integrated approach involves (i) text mining guided by RLIMS-P, a tool that identifies phosphorylation-related information in scientific literature; (ii) data mining from curated PPI databases; (iii) protein form and complex representation using the Protein Ontology (PRO); (iv) functional annotation using the Gene Ontology (GO); and (v) network visualization and analysis with Cytoscape. We use this framework to study the spindle checkpoint, the process that monitors the assembly of the mitotic spindle and blocks cell cycle progression at metaphase until all chromosomes have made bipolar spindle attachments. The phosphorylation networks we construct, centered on the human checkpoint kinase BUB1B (BubR1) and its yeast counterpart MAD3, offer a unique view of the spindle checkpoint that emphasizes biologically relevant phosphorylated forms, phosphorylation-state–specific PPIs and kinase–substrate relationships. Our approach for constructing protein phosphorylation networks can be applied to any biological process that is affected by phosphorylation.
Database URL: http://www.yeastgenome.org/
doi:10.1093/database/bat038
PMCID: PMC3675891  PMID: 23749465
11.  Structural and functional studies of S-adenosyl-L-methionine binding proteins: a ligand-centric approach 
Background
The post-genomic era poses several challenges. The biggest is the identification of biochemical function for protein sequences and structures resulting from genomic initiatives. Most sequences lack a characterized function and are annotated as hypothetical or uncharacterized. While homology-based methods are useful, and work well for sequences with sequence identities above 50%, they fail for sequences in the twilight zone (<30%) of sequence identity. For cases where sequence methods fail, structural approaches are often used, based on the premise that structure preserves function for longer evolutionary time-frames than sequence alone. It is now clear that no single method can be used successfully for functional inference. Given the growing need for functional assignments, we describe here a systematic new approach, designated ligand-centric, which is primarily based on analysis of ligand-bound/unbound structures in the PDB. Results of applying our approach to S-adenosyl-L-methionine (SAM) binding proteins are presented.
Results
Our analysis included 1,224 structures that belong to 172 unique families of the Protein Information Resource Superfamily system. Our ligand-centric approach was divided into four levels: residue, protein/domain, ligand, and family levels. The residue level included the identification of conserved binding site residues based on structure-guided sequence alignments of representative members of a family, and the identification of conserved structural motifs. The protein/domain level included structural classification of proteins, Pfam domains, domain architectures, and protein topologies. The ligand level included ligand conformations, ribose sugar puckering, and the identification of conserved ligand-atom interactions. The family level included phylogenetic analysis.
Conclusion
We found that SAM bound to a total of 18 different fold types (I-XVIII). We identified 4 new fold types and 11 additional topological arrangements of strands within the well-studied Rossmann fold Methyltransferases (MTases). This extends the existing structural classification of SAM binding proteins. A striking correlation between fold type and the conformation of the bound SAM (classified as types) was found across the 18 fold types. Several site-specific rules were created for the assignment of functional residues to families and proteins that do not have a bound SAM or a solved structure.
doi:10.1186/1472-6807-13-6
PMCID: PMC3662625  PMID: 23617634
13.  A comprehensive protein-centric ID mapping service for molecular data integration 
Bioinformatics  2011;27(8):1190-1191.
Motivation: Identifier (ID) mapping establishes links between various biological databases and is an essential first step for molecular data integration and functional annotation. ID mapping allows diverse molecular data on genes and proteins to be combined and mapped to functional pathways and ontologies. We have developed comprehensive protein-centric ID mapping services providing mappings for 90 IDs derived from databases on genes, proteins, pathways, diseases, structures, protein families, protein interaction, literature, ontologies, etc. The services are widely used and have been regularly updated since 2006.
Availability: www.uniprot.org/mappingandproteininformation-resource.org/pirwww/search/idmapping.shtml
Contact: huang@dbi.udel.edu
doi:10.1093/bioinformatics/btr101
PMCID: PMC3072559  PMID: 21478197
14.  Community annotation and bioinformatics workforce development in concert—Little Skate Genome Annotation Workshops and Jamborees 
Recent advances in high-throughput DNA sequencing technologies have equipped biologists with a powerful new set of tools for advancing research goals. The resulting flood of sequence data has made it critically important to train the next generation of scientists to handle the inherent bioinformatic challenges. The North East Bioinformatics Collaborative (NEBC) is undertaking the genome sequencing and annotation of the little skate (Leucoraja erinacea) to promote advancement of bioinformatics infrastructure in our region, with an emphasis on practical education to create a critical mass of informatically savvy life scientists. In support of the Little Skate Genome Project, the NEBC members have developed several annotation workshops and jamborees to provide training in genome sequencing, annotation and analysis. Acting as a nexus for both curation activities and dissemination of project data, a project web portal, SkateBase (http://skatebase.org) has been developed. As a case study to illustrate effective coupling of community annotation with workforce development, we report the results of the Mitochondrial Genome Annotation Jamborees organized to annotate the first completely assembled element of the Little Skate Genome Project, as a culminating experience for participants from our three prior annotation workshops. We are applying the physical/virtual infrastructure and lessons learned from these activities to enhance and streamline the genome annotation workflow, as we look toward our continuing efforts for larger-scale functional and structural community annotation of the L. erinacea genome.
doi:10.1093/database/bar064
PMCID: PMC3308154  PMID: 22434832
15.  Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells 
PLoS ONE  2011;6(6):e20410.
Background
Estrogen is a known growth promoter for estrogen receptor (ER)-positive breast cancer cells. Paradoxically, in breast cancer cells that have been chronically deprived of estrogen stimulation, re-introduction of the hormone can induce apoptosis.
Methodology/Principal Findings
Here, we sought to identify signaling networks that are triggered by estradiol (E2) in isogenic MCF-7 breast cancer cells that undergo apoptosis (MCF-7:5C) versus cells that proliferate upon exposure to E2 (MCF-7). The nuclear receptor co-activator AIB1 (Amplified in Breast Cancer-1) is known to be rate-limiting for E2-induced cell survival responses in MCF-7 cells and was found here to also be required for the induction of apoptosis by E2 in the MCF-7:5C cells. Proteins that interact with AIB1 as well as complexes that contain tyrosine phosphorylated proteins were isolated by immunoprecipitation and identified by mass spectrometry (MS) at baseline and after a brief exposure to E2 for two hours. Bioinformatic network analyses of the identified protein interactions were then used to analyze E2 signaling pathways that trigger apoptosis versus survival. Comparison of MS data with a computationally-predicted AIB1 interaction network showed that 26 proteins identified in this study are within this network, and are involved in signal transduction, transcription, cell cycle regulation and protein degradation.
Conclusions
G-protein-coupled receptors, PI3 kinase, Wnt and Notch signaling pathways were most strongly associated with E2-induced proliferation or apoptosis and are integrated here into a global AIB1 signaling network that controls qualitatively distinct responses to estrogen.
doi:10.1371/journal.pone.0020410
PMCID: PMC3124472  PMID: 21738574
16.  Representative Proteomes: A Stable, Scalable and Unbiased Proteome Set for Sequence Analysis and Functional Annotation 
PLoS ONE  2011;6(4):e18910.
The accelerating growth in the number of protein sequences taxes both the computational and manual resources needed to analyze them. One approach to dealing with this problem is to minimize the number of proteins subjected to such analysis in a way that minimizes loss of information. To this end we have developed a set of Representative Proteomes (RPs), each selected from a Representative Proteome Group (RPG) containing similar proteomes calculated based on co-membership in UniRef50 clusters. A Representative Proteome is the proteome that can best represent all the proteomes in its group in terms of the majority of the sequence space and information. RPs at 75%, 55%, 35% and 15% co-membership threshold (CMT) are provided to allow users to decrease or increase the granularity of the sequence space based on their requirements. We find that a CMT of 55% (RP55) most closely follows standard taxonomic classifications. Further analysis of this set reveals that sequence space is reduced by more than 80% relative to UniProtKB, while retaining both sequence diversity (over 95% of InterPro domains) and annotation information (93% of experimentally characterized proteins). All sets can be browsed and are available for sequence similarity searches and download at http://www.proteininformationresource.org/rps, while the set of 637 RPs determined using a 55% CMT are also available for text searches. Potential applications include sequence similarity searches, protein classification and targeted protein annotation and characterization.
doi:10.1371/journal.pone.0018910
PMCID: PMC3083393  PMID: 21556138
17.  The Protein Ontology: a structured representation of protein forms and complexes 
Nucleic Acids Research  2010;39(Database issue):D539-D545.
The Protein Ontology (PRO) provides a formal, logically-based classification of specific protein classes including structured representations of protein isoforms, variants and modified forms. Initially focused on proteins found in human, mouse and Escherichia coli, PRO now includes representations of protein complexes. The PRO Consortium works in concert with the developers of other biomedical ontologies and protein knowledge bases to provide the ability to formally organize and integrate representations of precise protein forms so as to enhance accessibility to results of protein research. PRO (http://pir.georgetown.edu/pro) is part of the Open Biomedical Ontology Foundry.
doi:10.1093/nar/gkq907
PMCID: PMC3013777  PMID: 20935045
18.  Phylogenomic Analysis of Marine Roseobacters 
PLoS ONE  2010;5(7):e11604.
Background
Members of the Roseobacter clade which play a key role in the biogeochemical cycles of the ocean are diverse and abundant, comprising 10–25% of the bacterioplankton in most marine surface waters. The rapid accumulation of whole-genome sequence data for the Roseobacter clade allows us to obtain a clearer picture of its evolution.
Methodology/Principal Findings
In this study about 1,200 likely orthologous protein families were identified from 17 Roseobacter bacteria genomes. Functional annotations for these genes are provided by iProClass. Phylogenetic trees were constructed for each gene using maximum likelihood (ML) and neighbor joining (NJ). Putative organismal phylogenetic trees were built with phylogenomic methods. These trees were compared and analyzed using principal coordinates analysis (PCoA), approximately unbiased (AU) and Shimodaira–Hasegawa (SH) tests. A core set of 694 genes with vertical descent signal that are resistant to horizontal gene transfer (HGT) is used to reconstruct a robust organismal phylogeny. In addition, we also discovered the most likely 109 HGT genes. The core set contains genes that encode ribosomal apparatus, ABC transporters and chaperones often found in the environmental metagenomic and metatranscriptomic data. These genes in the core set are spread out uniformly among the various functional classes and biological processes.
Conclusions/Significance
Here we report a new multigene-derived phylogenetic tree of the Roseobacter clade. Of particular interest is the HGT of eleven genes involved in vitamin B12 synthesis as well as key enzynmes for dimethylsulfoniopropionate (DMSP) degradation. These aquired genes are essential for the growth of Roseobacters and their eukaryotic partners.
doi:10.1371/journal.pone.0011604
PMCID: PMC2904699  PMID: 20657646
19.  Protein Bioinformatics Infrastructure for the Integration and Analysis of Multiple High-Throughput “omics” Data 
Advances in Bioinformatics  2010;2010:423589.
High-throughput “omics” technologies bring new opportunities for biological and biomedical researchers to ask complex questions and gain new scientific insights. However, the voluminous, complex, and context-dependent data being maintained in heterogeneous and distributed environments plus the lack of well-defined data standard and standardized nomenclature imposes a major challenge which requires advanced computational methods and bioinformatics infrastructures for integration, mining, visualization, and comparative analysis to facilitate data-driven hypothesis generation and biological knowledge discovery. In this paper, we present the challenges in high-throughput “omics” data integration and analysis, introduce a protein-centric approach for systems integration of large and heterogeneous high-throughput “omics” data including microarray, mass spectrometry, protein sequence, protein structure, and protein interaction data, and use scientific case study to illustrate how one can use varied “omics” data from different laboratories to make useful connections that could lead to new biological knowledge.
doi:10.1155/2010/423589
PMCID: PMC2847380  PMID: 20369061
20.  Systems Integration of Biodefense Omics Data for Analysis of Pathogen-Host Interactions and Identification of Potential Targets 
PLoS ONE  2009;4(9):e7162.
The NIAID (National Institute for Allergy and Infectious Diseases) Biodefense Proteomics program aims to identify targets for potential vaccines, therapeutics, and diagnostics for agents of concern in bioterrorism, including bacterial, parasitic, and viral pathogens. The program includes seven Proteomics Research Centers, generating diverse types of pathogen-host data, including mass spectrometry, microarray transcriptional profiles, protein interactions, protein structures and biological reagents. The Biodefense Resource Center (www.proteomicsresource.org) has developed a bioinformatics framework, employing a protein-centric approach to integrate and support mining and analysis of the large and heterogeneous data. Underlying this approach is a data warehouse with comprehensive protein + gene identifier and name mappings and annotations extracted from over 100 molecular databases. Value-added annotations are provided for key proteins from experimental findings using controlled vocabulary. The availability of pathogen and host omics data in an integrated framework allows global analysis of the data and comparisons across different experiments and organisms, as illustrated in several case studies presented here. (1) The identification of a hypothetical protein with differential gene and protein expressions in two host systems (mouse macrophage and human HeLa cells) infected by different bacterial (Bacillus anthracis and Salmonella typhimurium) and viral (orthopox) pathogens suggesting that this protein can be prioritized for additional analysis and functional characterization. (2) The analysis of a vaccinia-human protein interaction network supplemented with protein accumulation levels led to the identification of human Keratin, type II cytoskeletal 4 protein as a potential therapeutic target. (3) Comparison of complete genomes from pathogenic variants coupled with experimental information on complete proteomes allowed the identification and prioritization of ten potential diagnostic targets from Bacillus anthracis. The integrative analysis across data sets from multiple centers can reveal potential functional significance and hidden relationships between pathogen and host proteins, thereby providing a systems approach to basic understanding of pathogenicity and target identification.
doi:10.1371/journal.pone.0007162
PMCID: PMC2745575  PMID: 19779614
21.  Integrated Bioinformatics for Radiation-Induced Pathway Analysis from Proteomics and Microarray Data 
Functional analysis and interpretation of large-scale proteomics and gene expression data require effective use of bioinformatics tools and public knowledge resources coupled with expert-guided examination. An integrated bioinformatics approach was used to analyze cellular pathways in response to ionizing radiation. ATM, or ataxia-telangiectasia mutated , a serine-threonine protein kinase, plays critical roles in radiation responses, including cell cycle arrest and DNA repair. We analyzed radiation responsive pathways based on 2D-gel/MS proteomics and microarray gene expression data from fibroblasts expressing wild type or mutant ATM gene. The analysis showed that metabolism was significantly affected by radiation in an ATM dependent manner. In particular, purine metabolic pathways were differentially changed in the two cell lines. The expression of ribonucleoside-diphosphate reductase subunit M2 (RRM2) was increased in ATM-wild type cells at both mRNA and protein levels, but no changes were detected in ATM-mutated cells. Increased expression of p53 was observed 30min after irradiation of the ATM-wild type cells. These results suggest that RRM2 is a downstream target of the ATM-p53 pathway that mediates radiation-induced DNA repair. We demonstrated that the integrated bioinformatics approach facilitated pathway analysis, hypothesis generation and target gene/protein identification.
PMCID: PMC2603135  PMID: 19088860
bioinformatics; proteomics; radiation; purine metabolism; DNA repair; pathway and network
22.  Comparative Bioinformatics Analyses and Profiling of Lysosome-Related Organelle Proteomes 
Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for 7 lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.
doi:10.1016/j.ijms.2006.09.024
PMCID: PMC1828028  PMID: 17375895
23.  PIRSF Family Classification System for Protein Functional and Evolutionary Analysis 
The PIRSF protein classification system (http://pir.georgetown.edu/pirsf/) reflects evolutionary relationships of full-length proteins and domains. The primary PIRSF classification unit is the homeomorphic family, whose members are both homologous (evolved from a common ancestor) and homeomorphic (sharing full-length sequence similarity and a common domain architecture). PIRSF families are curated systematically based on literature review and integrative sequence and functional analysis, including sequence and structure similarity, domain architecture, functional association, genome context, and phyletic pattern. The results of classification and expert annotation are summarized in PIRSF family reports with graphical viewers for taxonomic distribution, domain architecture, family hierarchy, and multiple alignment and phylogenetic tree. The PIRSF system provides a comprehensive resource for bioinformatics analysis and comparative studies of protein function and evolution. Domain or fold-based searches allow identification of evolutionarily related protein families sharing domains or structural folds. Functional convergence and functional divergence are revealed by the relationships between protein classification and curated family functions. The taxonomic distribution allows the identification of lineage-specific or broadly conserved protein families and can reveal horizontal gene transfer. Here we demonstrate, with illustrative examples, how to use the web-based PIRSF system as a tool for functional and evolutionary studies of protein families.
PMCID: PMC2674652  PMID: 19455212
Domain architecture; Functional convergence; Functional divergence; Genome context; Protein family classification; Taxonomic distribution
24.  The Universal Protein Resource (UniProt): an expanding universe of protein information 
Nucleic Acids Research  2005;34(Database issue):D187-D191.
The Universal Protein Resource (UniProt) provides a central resource on protein sequences and functional annotation with three database components, each addressing a key need in protein bioinformatics. The UniProt Knowledgebase (UniProtKB), comprising the manually annotated UniProtKB/Swiss-Prot section and the automatically annotated UniProtKB/TrEMBL section, is the preeminent storehouse of protein annotation. The extensive cross-references, functional and feature annotations and literature-based evidence attribution enable scientists to analyse proteins and query across databases. The UniProt Reference Clusters (UniRef) speed similarity searches via sequence space compression by merging sequences that are 100% (UniRef100), 90% (UniRef90) or 50% (UniRef50) identical. Finally, the UniProt Archive (UniParc) stores all publicly available protein sequences, containing the history of sequence data with links to the source databases. UniProt databases continue to grow in size and in availability of information. Recent and upcoming changes to database contents, formats, controlled vocabularies and services are described. New download availability includes all major releases of UniProtKB, sequence collections by taxonomic division and complete proteomes. A bibliography mapping service has been added, and an ID mapping service will be available soon. UniProt databases can be accessed online at or downloaded at .
doi:10.1093/nar/gkj161
PMCID: PMC1347523  PMID: 16381842
25.  UniProt: the Universal Protein knowledgebase 
Nucleic Acids Research  2004;32(Database issue):D115-D119.
To provide the scientific community with a single, centralized, authoritative resource for protein sequences and functional information, the Swiss-Prot, TrEMBL and PIR protein database activities have united to form the Universal Protein Knowledgebase (UniProt) consortium. Our mission is to provide a comprehensive, fully classified, richly and accurately annotated protein sequence knowledgebase, with extensive cross-references and query interfaces. The central database will have two sections, corresponding to the familiar Swiss-Prot (fully manually curated entries) and TrEMBL (enriched with automated classification, annotation and extensive cross-references). For convenient sequence searches, UniProt also provides several non-redundant sequence databases. The UniProt NREF (UniRef) databases provide representative subsets of the knowledgebase suitable for efficient searching. The comprehensive UniProt Archive (UniParc) is updated daily from many public source databases. The UniProt databases can be accessed online (http://www.uniprot.org) or downloaded in several formats (ftp://ftp.uniprot.org/pub). The scientific community is encouraged to submit data for inclusion in UniProt.
doi:10.1093/nar/gkh131
PMCID: PMC308865  PMID: 14681372

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