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1.  The COMBREX Project: Design, Methodology, and Initial Results 
Anton, Brian P. | Chang, Yi-Chien | Brown, Peter | Choi, Han-Pil | Faller, Lina L. | Guleria, Jyotsna | Hu, Zhenjun | Klitgord, Niels | Levy-Moonshine, Ami | Maksad, Almaz | Mazumdar, Varun | McGettrick, Mark | Osmani, Lais | Pokrzywa, Revonda | Rachlin, John | Swaminathan, Rajeswari | Allen, Benjamin | Housman, Genevieve | Monahan, Caitlin | Rochussen, Krista | Tao, Kevin | Bhagwat, Ashok S. | Brenner, Steven E. | Columbus, Linda | de Crécy-Lagard, Valérie | Ferguson, Donald | Fomenkov, Alexey | Gadda, Giovanni | Morgan, Richard D. | Osterman, Andrei L. | Rodionov, Dmitry A. | Rodionova, Irina A. | Rudd, Kenneth E. | Söll, Dieter | Spain, James | Xu, Shuang-yong | Bateman, Alex | Blumenthal, Robert M. | Bollinger, J. Martin | Chang, Woo-Suk | Ferrer, Manuel | Friedberg, Iddo | Galperin, Michael Y. | Gobeill, Julien | Haft, Daniel | Hunt, John | Karp, Peter | Klimke, William | Krebs, Carsten | Macelis, Dana | Madupu, Ramana | Martin, Maria J. | Miller, Jeffrey H. | O'Donovan, Claire | Palsson, Bernhard | Ruch, Patrick | Setterdahl, Aaron | Sutton, Granger | Tate, John | Yakunin, Alexander | Tchigvintsev, Dmitri | Plata, Germán | Hu, Jie | Greiner, Russell | Horn, David | Sjölander, Kimmen | Salzberg, Steven L. | Vitkup, Dennis | Letovsky, Stanley | Segrè, Daniel | DeLisi, Charles | Roberts, Richard J. | Steffen, Martin | Kasif, Simon
PLoS Biology  2013;11(8):e1001638.
Experimental data exists for only a vanishingly small fraction of sequenced microbial genes. This community page discusses the progress made by the COMBREX project to address this important issue using both computational and experimental resources.
PMCID: PMC3754883  PMID: 24013487
2.  VisANT 4.0: Integrative network platform to connect genes, drugs, diseases and therapies 
Nucleic Acids Research  2013;41(Web Server issue):W225-W231.
With the rapid accumulation of our knowledge on diseases, disease-related genes and drug targets, network-based analysis plays an increasingly important role in systems biology, systems pharmacology and translational science. The new release of VisANT aims to provide new functions to facilitate the convenient network analysis of diseases, therapies, genes and drugs. With improved understanding of the mechanisms of complex diseases and drug actions through network analysis, novel drug methods (e.g., drug repositioning, multi-target drug and combination therapy) can be designed. More specifically, the new update includes (i) integrated search and navigation of disease and drug hierarchies; (ii) integrated disease–gene, therapy–drug and drug–target association to aid the network construction and filtering; (iii) annotation of genes/drugs using disease/therapy information; (iv) prediction of associated diseases/therapies for a given set of genes/drugs using enrichment analysis; (v) network transformation to support construction of versatile network of drugs, genes, diseases and therapies; (vi) enhanced user interface using docking windows to allow easy customization of node and edge properties with build-in legend node to distinguish different node type. VisANT is freely available at:
PMCID: PMC3692070  PMID: 23716640
3.  Gene set enrichment analysis: performance evaluation and usage guidelines 
Briefings in Bioinformatics  2011;13(3):281-291.
A central goal of biology is understanding and describing the molecular basis of plasticity: the sets of genes that are combinatorially selected by exogenous and endogenous environmental changes, and the relations among the genes. The most viable current approach to this problem consists of determining whether sets of genes are connected by some common theme, e.g. genes from the same pathway are overrepresented among those whose differential expression in response to a perturbation is most pronounced. There are many approaches to this problem, and the results they produce show a fair amount of dispersion, but they all fall within a common framework consisting of a few basic components. We critically review these components, suggest best practices for carrying out each step, and propose a voting method for meeting the challenge of assessing different methods on a large number of experimental data sets in the absence of a gold standard.
PMCID: PMC3357488  PMID: 21900207
gene set enrichment analysis; pathway enrichment analysis; expression analysis; GSEA; PWEA; performance evaluation; controlled mutual coverage; CMC
4.  The NIH National Center for Integrative Biomedical Informatics (NCIBI) 
The National Center for Integrative and Biomedical Informatics (NCIBI) is one of the eight NCBCs. NCIBI supports information access and data analysis for biomedical researchers, enabling them to build computational and knowledge models of biological systems to address the Driving Biological Problems (DBPs). The NCIBI DBPs have included prostate cancer progression, organ-specific complications of type 1 and 2 diabetes, bipolar disorder, and metabolic analysis of obesity syndrome. Collaborating with these and other partners, NCIBI has developed a series of software tools for exploratory analysis, concept visualization, and literature searches, as well as core database and web services resources. Many of our training and outreach initiatives have been in collaboration with the Research Centers at Minority Institutions (RCMI), integrating NCIBI and RCMI faculty and students, culminating each year in an annual workshop. Our future directions include focusing on the TranSMART data sharing and analysis initiative.
PMCID: PMC3277628  PMID: 22101971
Integrative; bioinformatics; computational biology; biomedical informatics; biomedical informatics; genomics; national center
5.  Pathway-based classification of cancer subtypes 
Biology Direct  2012;7:21.
Molecular markers based on gene expression profiles have been used in experimental and clinical settings to distinguish cancerous tumors in stage, grade, survival time, metastasis, and drug sensitivity. However, most significant gene markers are unstable (not reproducible) among data sets. We introduce a standardized method for representing cancer markers as 2-level hierarchical feature vectors, with a basic gene level as well as a second level of (more stable) pathway markers, for the purpose of discriminating cancer subtypes. This extends standard gene expression arrays with new pathway-level activation features obtained directly from off-the-shelf gene set enrichment algorithms such as GSEA. Such so-called pathway-based expression arrays are significantly more reproducible across datasets. Such reproducibility will be important for clinical usefulness of genomic markers, and augment currently accepted cancer classification protocols.
The present method produced more stable (reproducible) pathway-based markers for discriminating breast cancer metastasis and ovarian cancer survival time. Between two datasets for breast cancer metastasis, the intersection of standard significant gene biomarkers totaled 7.47% of selected genes, compared to 17.65% using pathway-based markers; the corresponding percentages for ovarian cancer datasets were 20.65% and 33.33% respectively. Three pathways, consisting of Type_1_diabetes mellitus, Cytokine-cytokine_receptor_interaction and Hedgehog_signaling (all previously implicated in cancer), are enriched in both the ovarian long survival and breast non-metastasis groups. In addition, integrating pathway and gene information, we identified five (ID4, ANXA4, CXCL9, MYLK, FBXL7) and six (SQLE, E2F1, PTTG1, TSTA3, BUB1B, MAD2L1) known cancer genes significant for ovarian and breast cancer respectively.
Standardizing the analysis of genomic data in the process of cancer staging, classification and analysis is important as it has implications for both pre-clinical as well as clinical studies. The paradigm of diagnosis and prediction using pathway-based biomarkers as features can be an important part of the process of biomarker-based cancer analysis, and the resulting canonical (clinically reproducible) biomarkers can be important in standardizing genomic data. We expect that identification of such canonical biomarkers will improve clinical utility of high-throughput datasets for diagnostic and prognostic applications.
This article was reviewed by John McDonald (nominated by I. King Jordon), Eugene Koonin, Nathan Bowen (nominated by I. King Jordon), and Ekaterina Kotelnikova (nominated by Mikhail Gelfand).
PMCID: PMC3485163  PMID: 22759382
Classification; Pathway; Cancer subtypes; Ovarian cancer; Breast cancer; Gene set enrichment analysis
6.  Correlation set analysis: detecting active regulators in disease populations using prior causal knowledge 
BMC Bioinformatics  2012;13:46.
Identification of active causal regulators is a crucial problem in understanding mechanism of diseases or finding drug targets. Methods that infer causal regulators directly from primary data have been proposed and successfully validated in some cases. These methods necessarily require very large sample sizes or a mix of different data types. Recent studies have shown that prior biological knowledge can successfully boost a method's ability to find regulators.
We present a simple data-driven method, Correlation Set Analysis (CSA), for comprehensively detecting active regulators in disease populations by integrating co-expression analysis and a specific type of literature-derived causal relationships. Instead of investigating the co-expression level between regulators and their regulatees, we focus on coherence of regulatees of a regulator. Using simulated datasets we show that our method performs very well at recovering even weak regulatory relationships with a low false discovery rate. Using three separate real biological datasets we were able to recover well known and as yet undescribed, active regulators for each disease population. The results are represented as a rank-ordered list of regulators, and reveals both single and higher-order regulatory relationships.
CSA is an intuitive data-driven way of selecting directed perturbation experiments that are relevant to a disease population of interest and represent a starting point for further investigation. Our findings demonstrate that combining co-expression analysis on regulatee sets with a literature-derived network can successfully identify causal regulators and help develop possible hypothesis to explain disease progression.
PMCID: PMC3382432  PMID: 22443377
7.  Using Functional Signatures to Identify Repositioned Drugs for Breast, Myelogenous Leukemia and Prostate Cancer 
PLoS Computational Biology  2012;8(2):e1002347.
The cost and time to develop a drug continues to be a major barrier to widespread distribution of medication. Although the genomic revolution appears to have had little impact on this problem, and might even have exacerbated it because of the flood of additional and usually ineffective leads, the emergence of high throughput resources promises the possibility of rapid, reliable and systematic identification of approved drugs for originally unintended uses. In this paper we develop and apply a method for identifying such repositioned drug candidates against breast cancer, myelogenous leukemia and prostate cancer by looking for inverse correlations between the most perturbed gene expression levels in human cancer tissue and the most perturbed expression levels induced by bioactive compounds. The method uses variable gene signatures to identify bioactive compounds that modulate a given disease. This is in contrast to previous methods that use small and fixed signatures. This strategy is based on the observation that diseases stem from failed/modified cellular functions, irrespective of the particular genes that contribute to the function, i.e., this strategy targets the functional signatures for a given cancer. This function-based strategy broadens the search space for the effective drugs with an impressive hit rate. Among the 79, 94 and 88 candidate drugs for breast cancer, myelogenous leukemia and prostate cancer, 32%, 13% and 17% respectively are either FDA-approved/in-clinical-trial drugs, or drugs with suggestive literature evidences, with an FDR of 0.01. These findings indicate that the method presented here could lead to a substantial increase in efficiency in drug discovery and development, and has potential application for the personalized medicine.
Author Summary
The effective drug of a given disease is aimed to bring abnormal functions associated with disease back to the normal state. Using expression profile as the surrogate marker of the cellular function, we introduce a novel procedure to identify candidate therapeutics by searching for those bioactive compounds that either down-regulate abnormally over-expressed genes, or up-regulate those that are abnormally under-expressed. We show that the approach detects a pool of plausible candidates as repositioning/new drugs. In contrast to previous studies, our approach uses a variable big number of genes and/or gene combinations as a representation of functional signatures to identify bioactive compounds that modulate a given disease, irrespective of the particular genes that contribute to the cellular functions; therefore it covers potential drugs with heterogeneous properties. The method may also have potential application for the personalized medicine.
PMCID: PMC3276504  PMID: 22346740
8.  Combinations of newly confirmed Glioma-Associated loci link regions on chromosomes 1 and 9 to increased disease risk 
BMC Medical Genomics  2011;4:63.
Glioblastoma multiforme (GBM) tends to occur between the ages of 45 and 70. This relatively early onset and its poor prognosis make the impact of GBM on public health far greater than would be suggested by its relatively low frequency. Tissue and blood samples have now been collected for a number of populations, and predisposing alleles have been sought by several different genome-wide association (GWA) studies. The Cancer Genome Atlas (TCGA) at NIH has also collected a considerable amount of data. Because of the low concordance between the results obtained using different populations, only 14 predisposing single nucleotide polymorphism (SNP) candidates in five genomic regions have been replicated in two or more studies. The purpose of this paper is to present an improved approach to biomarker identification.
Association analysis was performed with control of population stratifications using the EIGENSTRAT package, under the null hypothesis of "no association between GBM and control SNP genotypes," based on an additive inheritance model. Genes that are strongly correlated with identified SNPs were determined by linkage disequilibrium (LD) or expression quantitative trait locus (eQTL) analysis. A new approach that combines meta-analysis and pathway enrichment analysis identified additional genes.
(i) A meta-analysis of SNP data from TCGA and the Adult Glioma Study identifies 12 predisposing SNP candidates, seven of which are reported for the first time. These SNPs fall in five genomic regions (5p15.33, 9p21.3, 1p21.2, 3q26.2 and 7p15.3), three of which have not been previously reported. (ii) 25 genes are strongly correlated with these 12 SNPs, eight of which are known to be cancer-associated. (iii) The relative risk for GBM is highest for risk allele combinations on chromosomes 1 and 9. (iv) A combined meta-analysis/pathway analysis identified an additional four genes. All of these have been identified as cancer-related, but have not been previously associated with glioma. (v) Some SNPs that do not occur reproducibly across populations are in reproducible (invariant) pathways, suggesting that they affect the same biological process, and that population discordance can be partially resolved by evaluating processes rather than genes.
We have uncovered 29 glioma-associated gene candidates; 12 of them known to be cancer related (p = 1. 4 × 10-6), providing additional statistical support for the relevance of the new candidates. This additional information on risk loci is potentially important for identifying Caucasian individuals at risk for glioma, and for assessing relative risk.
PMCID: PMC3212919  PMID: 21827660
9.  Phenotypic connections in surprising places 
Genome Biology  2010;11(4):116.
Connections have been revealed between very different human diseases using phenotype associations in other species
Surprising correlations between human disease phenotypes are emerging. Recent work now reveals startling phenotype connections between species, which could provide new disease models.
PMCID: PMC2884535  PMID: 20423531
10.  Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches 
Biosensors & bioelectronics  2010;25(7):1789-1795.
A novel method is proposed for direct detection of DNA hybridization on microarrays. Optical interferometry is used for label-free sensing of biomolecular accumulation on glass surfaces, enabling dynamic detection of interactions. Capabilities of the presented method are demonstrated by high-throughput sensing of solid-phase hybridization of oligonucleotides. Hybridization of surface immobilized probes with 20 base pair-long target oligonucleotides was detected by comparing the label-free microarray images taken before and after hybridization. Through dynamic data acquisition during denaturation by washing the sample with low ionic concentration buffer, melting of duplexes with a single nucleotide mismatch was distinguished from perfectly matching duplexes with high confidence interval (>97%). The presented technique is simple, robust, and accurate, and eliminates the need of using labels or secondary reagents to monitor the oligonucleotide hybridization.
PMCID: PMC2824047  PMID: 20097056
11.  COMBREX: a project to accelerate the functional annotation of prokaryotic genomes 
Nucleic Acids Research  2010;39(Database issue):D11-D14.
COMBREX ( is a project to increase the speed of the functional annotation of new bacterial and archaeal genomes. It consists of a database of functional predictions produced by computational biologists and a mechanism for experimental biochemists to bid for the validation of those predictions. Small grants are available to support successful bids.
PMCID: PMC3013729  PMID: 21097892
12.  Identification of functional modules that correlate with phenotypic difference: the influence of network topology 
Genome Biology  2010;11(2):R23.
A gene set enrichment analysis method for including network topology in the identification of genes involved in phenotypic alterations is described. Classifications: Genome studies, Methods
One of the important challenges to post-genomic biology is relating observed phenotypic alterations to the underlying collective alterations in genes. Current inferential methods, however, invariably omit large bodies of information on the relationships between genes. We present a method that takes account of such information - expressed in terms of the topology of a correlation network - and we apply the method in the context of current procedures for gene set enrichment analysis.
PMCID: PMC2872883  PMID: 20187943
13.  Resonant Cavity Imaging: A Means Toward High-Throughput Label-Free Protein Detection 
The resonant cavity imaging biosensor (RCIB) is an optical technique for detecting molecular binding interactions label free at many locations in parallel that employs an optical resonant cavity for high sensitivity. Near-infrared light centered at 1512.5 nm couples resonantly through a Fabry–Perot cavity constructed from dielectric reflectors (Si/SiO2), one of which serves as the binding surface. As the wavelength is swept using a tunable laser, a near-infrared digital camera monitors cavity transmittance at each pixel. A wavelength shift in the local resonant response of the optical cavity indicates binding. Positioning the sensing surface with respect to the standing wave pattern of the electric field within the cavity controls the sensitivity with which the presence of bound molecules is detected. Transmitted intensity at thousands of pixel locations is recorded simultaneously in a 10 s, 5 nm scan. An initial proof-of-principle setup has been constructed. A test sample was fabricated with 25, 100-μm wide square features, each with a different density of 1-μm square depressions etched 12 nm into the SiO2 surface. The average depth of each etched region was found with 0.05 nm rms precision. In a second test, avidin, bound selectively to biotin conjugated bovine serum albumin, was detected.
PMCID: PMC2759719  PMID: 19823593
Biochemistry; chemical transducers; optical resonance
14.  VisANT: an integrative framework for networks in systems biology 
Briefings in bioinformatics  2008;9(4):317-325.
The essence of a living cell is adaptation to a changing environment, and a central goal of modern cell biology is to understand adaptive change under normal and pathological conditions. Because the number of components is large, and processes and conditions are many, visual tools are useful in providing an overview of relations that would otherwise be far more difficult to assimilate. Historically, representations were static pictures, with genes and 10 proteins represented as nodes, and known or inferred correlations between them (links) represented by various kinds of lines. The modern challenge is to capture functional hierarchies and adaptation to environmental change, and to discover pathways and processes embedded in known data, but not currently recognizable. Among the tools being developed to meet this challenge is VisANT (freely available at which integrates, mines and displays hierarchical information. Challenges to integrating modeling (discrete or continuous) and simulation capabilities into such visual mining software are briefly discussed.
PMCID: PMC2743399  PMID: 18463131
network; integration; systems biology; metagraph; visualization
15.  Genome-wide prioritization of disease genes and identification of disease-disease associations from an integrated human functional linkage network 
Genome Biology  2009;10(9):R91.
An evidence-weighted functional-linkage network of human genes reveals associations among diseases that share no known disease genes and have dissimilar phenotypes
We integrate 16 genomic features to construct an evidence-weighted functional-linkage network comprising 21,657 human genes. The functional-linkage network is used to prioritize candidate genes for 110 diseases, and to reliably disclose hidden associations between disease pairs having dissimilar phenotypes, such as hypercholesterolemia and Alzheimer's disease. Many of these disease-disease associations are supported by epidemiology, but with no previous genetic basis. Such associations can drive novel hypotheses on molecular mechanisms of diseases and therapies.
PMCID: PMC2768980  PMID: 19728866
16.  VisANT 3.5: multi-scale network visualization, analysis and inference based on the gene ontology 
Nucleic Acids Research  2009;37(Web Server issue):W115-W121.
Despite its wide usage in biological databases and applications, the role of the gene ontology (GO) in network analysis is usually limited to functional annotation of genes or gene sets with auxiliary information on correlations ignored. Here, we report on new capabilities of VisANT—an integrative software platform for the visualization, mining, analysis and modeling of the biological networks—which extend the application of GO in network visualization, analysis and inference. The new VisANT functions can be classified into three categories. (i) Visualization: a new tree-based browser allows visualization of GO hierarchies. GO terms can be easily dropped into the network to group genes annotated under the term, thereby integrating the hierarchical ontology with the network. This facilitates multi-scale visualization and analysis. (ii) Flexible annotation schema: in addition to conventional methods for annotating network nodes with the most specific functional descriptions available, VisANT also provides functions to annotate genes at any customized level of abstraction. (iii) Finding over-represented GO terms and expression-enriched GO modules: two new algorithms have been implemented as VisANT plugins. One detects over-represented GO annotations in any given sub-network and the other finds the GO categories that are enriched in a specified phenotype or perturbed dataset. Both algorithms take account of network topology (i.e. correlations between genes based on various sources of evidence). VisANT is freely available at
PMCID: PMC2703932  PMID: 19465394
17.  In silico regulatory analysis for exploring human disease progression 
Biology Direct  2008;3:24.
An important goal in bioinformatics is to unravel the network of transcription factors (TFs) and their targets. This is important in the human genome, where many TFs are involved in disease progression. Here, classification methods are applied to identify new targets for 152 transcriptional regulators using publicly-available targets as training examples. Three types of sequence information are used: composition, conservation, and overrepresentation.
Starting with 8817 TF-target interactions we predict an additional 9333 targets for 152 TFs. Randomized classifiers make few predictions (~2/18660) indicating that our predictions for many TFs are significantly enriched for true targets. An enrichment score is calculated and used to filter new predictions.
Two case-studies for the TFs OCT4 and WT1 illustrate the usefulness of our predictions:
• Many predicted OCT4 targets fall into the Wnt-pathway. This is consistent with known biology as OCT4 is developmentally related and Wnt pathway plays a role in early development.
• Beginning with 15 known targets, 354 predictions are made for WT1. WT1 has a role in formation of Wilms' tumor. Chromosomal regions previously implicated in Wilms' tumor by cytological evidence are statistically enriched in predicted WT1 targets. These findings may shed light on Wilms' tumor progression, suggesting that the tumor progresses either by loss of WT1 or by loss of regions harbouring its targets.
• Targets of WT1 are statistically enriched for cancer related functions including metastasis and apoptosis. Among new targets are BAX and PDE4B, which may help mediate the established anti-apoptotic effects of WT1.
• Of the thirteen TFs found which co-regulate genes with WT1 (p ≤ 0.02), 8 have been previously implicated in cancer. The regulatory-network for WT1 targets in genomic regions relevant to Wilms' tumor is provided.
We have assembled a set of features for the targets of human TFs and used them to develop classifiers for the determination of new regulatory targets. Many predicted targets are consistent with the known biology of their regulators, and new targets for the Wilms' tumor regulator, WT1, are proposed. We speculate that Wilms' tumor development is mediated by chromosomal rearrangements in the location of WT1 targets.
This article was reviewed by Trey Ideker, Vladimir A. Kuznetsov(nominated by Frank Eisenhaber), and Tzachi Pilpel.
PMCID: PMC2464594  PMID: 18564415
18.  Classifying transcription factor targets and discovering relevant biological features 
Biology Direct  2008;3:22.
An important goal in post-genomic research is discovering the network of interactions between transcription factors (TFs) and the genes they regulate. We have previously reported the development of a supervised-learning approach to TF target identification, and used it to predict targets of 104 transcription factors in yeast. We now include a new sequence conservation measure, expand our predictions to include 59 new TFs, introduce a web-server, and implement an improved ranking method to reveal the biological features contributing to regulation. The classifiers combine 8 genomic datasets covering a broad range of measurements including sequence conservation, sequence overrepresentation, gene expression, and DNA structural properties.
Principal Findings
(1) Application of the method yields an amplification of information about yeast regulators. The ratio of total targets to previously known targets is greater than 2 for 11 TFs, with several having larger gains: Ash1(4), Ino2(2.6), Yaf1(2.4), and Yap6(2.4).
(2) Many predicted targets for TFs match well with the known biology of their regulators. As a case study we discuss the regulator Swi6, presenting evidence that it may be important in the DNA damage response, and that the previously uncharacterized gene YMR279C plays a role in DNA damage response and perhaps in cell-cycle progression.
(3) A procedure based on recursive-feature-elimination is able to uncover from the large initial data sets those features that best distinguish targets for any TF, providing clues relevant to its biology. An analysis of Swi6 suggests a possible role in lipid metabolism, and more specifically in metabolism of ceramide, a bioactive lipid currently being investigated for anti-cancer properties.
(4) An analysis of global network properties highlights the transcriptional network hubs; the factors which control the most genes and the genes which are bound by the largest set of regulators. Cell-cycle and growth related regulators dominate the former; genes involved in carbon metabolism and energy generation dominate the latter.
Postprocessing of regulatory-classifier results can provide high quality predictions, and feature ranking strategies can deliver insight into the regulatory functions of TFs. Predictions are available at an online web-server, including the full transcriptional network, which can be analyzed using VisAnt network analysis suite.
This article was reviewed by Igor Jouline, Todd Mockler(nominated by Valerian Dolja), and Sandor Pongor.
PMCID: PMC2441612  PMID: 18513408
19.  High-precision high-coverage functional inference from integrated data sources 
BMC Bioinformatics  2008;9:119.
Information obtained from diverse data sources can be combined in a principled manner using various machine learning methods to increase the reliability and range of knowledge about protein function. The result is a weighted functional linkage network (FLN) in which linked neighbors share at least one function with high probability. Precision is, however, low. Aiming to provide precise functional annotation for as many proteins as possible, we explore and propose a two-step framework for functional annotation (1) construction of a high-coverage and reliable FLN via machine learning techniques (2) development of a decision rule for the constructed FLN to optimize functional annotation.
We first apply this framework to Saccharomyces cerevisiae. In the first step, we demonstrate that four commonly used machine learning methods, Linear SVM, Linear Discriminant Analysis, Naïve Bayes, and Neural Network, all combine heterogeneous data to produce reliable and high-coverage FLNs, in which the linkage weight more accurately estimates functional coupling of linked proteins than use individual data sources alone. In the second step, empirical tuning of an adjustable decision rule on the constructed FLN reveals that basing annotation on maximum edge weight results in the most precise annotation at high coverages. In particular at low coverage all rules evaluated perform comparably. At coverage above approximately 50%, however, they diverge rapidly. At full coverage, the maximum weight decision rule still has a precision of approximately 70%, whereas for other methods, precision ranges from a high of slightly more than 30%, down to 3%. In addition, a scoring scheme to estimate the precisions of individual predictions is also provided. Finally, tests of the robustness of the framework indicate that our framework can be successfully applied to less studied organisms.
We provide a general two-step function-annotation framework, and show that high coverage, high precision annotations can be achieved by constructing a high-coverage and reliable FLN via data integration followed by applying a maximum weight decision rule.
PMCID: PMC2292694  PMID: 18298847
20.  Portraits of breast cancer progression 
BMC Bioinformatics  2007;8:291.
Clustering analysis of microarray data is often criticized for giving ambiguous results because of sensitivity to data perturbation or clustering techniques used. In this paper, we describe a new method based on principal component analysis and ensemble consensus clustering that avoids these problems.
We illustrate the method on a public microarray dataset from 36 breast cancer patients of whom 31 were diagnosed with at least two of three pathological stages of disease (atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Our method identifies an optimum set of genes and divides the samples into stable clusters which correlate with clinical classification into Luminal, Basal-like and Her2+ subtypes. Our analysis reveals a hierarchical portrait of breast cancer progression and identifies genes and pathways for each stage, grade and subtype. An intriguing observation is that the disease phenotype is distinguishable in ADH and progresses along distinct pathways for each subtype. The genetic signature for disease heterogeneity across subtypes is greater than the heterogeneity of progression from DCIS to IDC within a subtype, suggesting that the disease subtypes have distinct progression pathways.
Our method identifies six disease subtype and one normal clusters. The first split separates the normal samples from the cancer samples. Next, the cancer cluster splits into low grade (pathological grades 1 and 2) and high grade (pathological grades 2 and 3) while the normal cluster is unchanged. Further, the low grade cluster splits into two subclusters and the high grade cluster into four. The final six disease clusters are mapped into one Luminal A, three Luminal B, one Basal-like and one Her2+.
We confirm that the cancer phenotype can be identified in early stage because the genes altered in this stage progressively alter further as the disease progresses through DCIS into IDC. We identify six subtypes of disease which have distinct genetic signatures and remain separated in the clustering hierarchy. Our findings suggest that the heterogeneity of disease across subtypes is higher than the heterogeneity of the disease progression within a subtype, indicating that the subtypes are in fact distinct diseases.
PMCID: PMC1978212  PMID: 17683614
21.  VisANT 3.0: new modules for pathway visualization, editing, prediction and construction 
Nucleic Acids Research  2007;35(Web Server issue):W625-W632.
With the integration of the KEGG and Predictome databases as well as two search engines for coexpressed genes/proteins using data sets obtained from the Stanford Microarray Database (SMD) and Gene Expression Omnibus (GEO) database, VisANT 3.0 supports exploratory pathway analysis, which includes multi-scale visualization of multiple pathways, editing and annotating pathways using a KEGG compatible visual notation and visualization of expression data in the context of pathways. Expression levels are represented either by color intensity or by nodes with an embedded expression profile. Multiple experiments can be navigated or animated. Known KEGG pathways can be enriched by querying either coexpressed components of known pathway members or proteins with known physical interactions. Predicted pathways for genes/proteins with unknown functions can be inferred from coexpression or physical interaction data. Pathways produced in VisANT can be saved as computer-readable XML format (VisML), graphic images or high-resolution Scalable Vector Graphics (SVG). Pathways in the format of VisML can be securely shared within an interested group or published online using a simple Web link. VisANT is freely available at
PMCID: PMC1933155  PMID: 17586824
22.  Binding Site Graphs: A New Graph Theoretical Framework for Prediction of Transcription Factor Binding Sites 
PLoS Computational Biology  2007;3(5):e90.
Computational prediction of nucleotide binding specificity for transcription factors remains a fundamental and largely unsolved problem. Determination of binding positions is a prerequisite for research in gene regulation, a major mechanism controlling phenotypic diversity. Furthermore, an accurate determination of binding specificities from high-throughput data sources is necessary to realize the full potential of systems biology. Unfortunately, recently performed independent evaluation showed that more than half the predictions from most widely used algorithms are false. We introduce a graph-theoretical framework to describe local sequence similarity as the pair-wise distances between nucleotides in promoter sequences, and hypothesize that densely connected subgraphs are indicative of transcription factor binding sites. Using a well-established sampling algorithm coupled with simple clustering and scoring schemes, we identify sets of closely related nucleotides and test those for known TF binding activity. Using an independent benchmark, we find our algorithm predicts yeast binding motifs considerably better than currently available techniques and without manual curation. Importantly, we reduce the number of false positive predictions in yeast to less than 30%. We also develop a framework to evaluate the statistical significance of our motif predictions. We show that our approach is robust to the choice of input promoters, and thus can be used in the context of predicting binding positions from noisy experimental data. We apply our method to identify binding sites using data from genome scale ChIP–chip experiments. Results from these experiments are publicly available at The graphical framework developed here may be useful when combining predictions from numerous computational and experimental measures. Finally, we discuss how our algorithm can be used to improve the sensitivity of computational predictions of transcription factor binding specificities.
Author Summary
A historically difficult problem in computational biology is the identification of transcription factor binding sites (TFBS) in the promoters of co-regulated genes. With increasing emphasis on research in transcriptional regulation, this problem is also uniquely relevant to emerging results from recent experiments in high-throughput and systems biology. Despite extensive research in the area, recent evaluations of previously published techniques show much room for improvement. In this paper, we introduce a fundamentally new approach to the identification of TFBS. First, we start by representing nucleotides in promoters as an undirected, weighted graph. Given this representation of a binding site graph (BSG), we employ relatively simple graph clustering techniques to identify functional TFBS. We show that BSG predictions significantly outperform all previously evaluated methods in nearly every performance measure using a standardized assessment benchmark. We also find that this approach is more robust than traditional Gibbs sampling to selection of input promoters, and thus more likely to perform well under noisy experimental conditions. Finally, BSGs are very good at predicting specificity determining nucleotides. Using BSG predictions, we were able to confirm recent experimental results on binding specificity of E-box TFs CBF1 and PHO4 and predict novel specificity determining nucleotides for TYE7.
PMCID: PMC1866359  PMID: 17500587
23.  Identification and Characterization of Renal Cell Carcinoma Gene Markers 
Cancer Informatics  2007;3:65-92.
Microarray gene expression profiling has been used to distinguish histological subtypes of renal cell carcinoma (RCC), and consequently to identify specific tumor markers. The analytical procedures currently in use find sets of genes whose average differential expression across the two categories differ significantly. In general each of the markers thus identified does not distinguish tumor from normal with 100% accuracy, although the group as a whole might be able to do so. For the purpose of developing a widely used economically viable diagnostic signature, however, large groups of genes are not likely to be useful. Here we use two different methods, one a support vector machine variant, and the other an exhaustive search, to reanalyze data previously generated in our Lab (Lenburg et al. 2003). We identify 158 genes, each having an expression level that is higher (lower) in every tumor sample than in any normal sample, and each having a minimum differential expression across the two categories at a significance of 0.01. The set is highly enriched in cancer related genes (p = 1.6 × 10−12), containing 43 genes previously associated with either RCC or other types of cancer. Many of the biomarkers appear to be associated with the central alterations known to be required for cancer transformation. These include the oncogenes JAZF1, AXL, ABL2; tumor suppressors RASD1, PTPRO, TFAP2A, CDKN1C; and genes involved in proteolysis or cell-adhesion such as WASF2, and PAPPA.
PMCID: PMC2675843  PMID: 19455236
Cancer diagnosis; biomarker identification; microarray analysis; Renal cell carcinoma
24.  Machine learning for regulatory analysis and transcription factor target prediction in yeast 
Systems and Synthetic Biology  2006;1(1):25-46.
High throughput technologies, including array-based chromatin immunoprecipitation, have rapidly increased our knowledge of transcriptional maps—the identity and location of regulatory binding sites within genomes. Still, the full identification of sites, even in lower eukaryotes, remains largely incomplete. In this paper we develop a supervised learning approach to site identification using support vector machines (SVMs) to combine 26 different data types. A comparison with the standard approach to site identification using position specific scoring matrices (PSSMs) for a set of 104 Saccharomyces cerevisiae regulators indicates that our SVM-based target classification is more sensitive (73 vs. 20%) when specificity and positive predictive value are the same. We have applied our SVM classifier for each transcriptional regulator to all promoters in the yeast genome to obtain thousands of new targets, which are currently being analyzed and refined to limit the risk of classifier over-fitting. For the purpose of illustration we discuss several results, including biochemical pathway predictions for Gcn4 and Rap1. For both transcription factors SVM predictions match well with the known biology of control mechanisms, and possible new roles for these factors are suggested, such as a function for Rap1 in regulating fermentative growth. We also examine the promoter melting temperature curves for the targets of YJR060W, and show that targets of this TF have potentially unique physical properties which distinguish them from other genes. The SVM output automatically provides the means to rank dataset features to identify important biological elements. We use this property to rank classifying k-mers, thereby reconstructing known binding sites for several TFs, and to rank expression experiments, determining the conditions under which Fhl1, the factor responsible for expression of ribosomal protein genes, is active. We can see that targets of Fhl1 are differentially expressed in the chosen conditions as compared to the expression of average and negative set genes. SVM-based classifiers provide a robust framework for analysis of regulatory networks. Processing of classifier outputs can provide high quality predictions and biological insight into functions of particular transcription factors. Future work on this method will focus on increasing the accuracy and quality of predictions using feature reduction and clustering strategies. Since predictions have been made on only 104 TFs in yeast, new classifiers will be built for the remaining 100 factors which have available binding data.
Electronic Supplementary Material
Supplementary material is available in the online version of this article at and is accessible for authorized users.
PMCID: PMC2533145  PMID: 19003435
Transcription factor; SVM; Machine learning
25.  Positional clustering improves computational binding site detection and identifies novel cis-regulatory sites in mammalian GABAA receptor subunit genes 
Nucleic Acids Research  2007;35(3):e20.
Understanding transcription factor (TF) mediated control of gene expression remains a major challenge at the interface of computational and experimental biology. Computational techniques predicting TF-binding site specificity are frequently unreliable. On the other hand, comprehensive experimental validation is difficult and time consuming. We introduce a simple strategy that dramatically improves robustness and accuracy of computational binding site prediction. First, we evaluate the rate of recurrence of computational TFBS predictions by commonly used sampling procedures. We find that the vast majority of results are biologically meaningless. However clustering results based on nucleotide position improves predictive power. Additionally, we find that positional clustering increases robustness to long or imperfectly selected input sequences. Positional clustering can also be used as a mechanism to integrate results from multiple sampling approaches for improvements in accuracy over each one alone. Finally, we predict and validate regulatory sequences partially responsible for transcriptional control of the mammalian type A γ-aminobutyric acid receptor (GABAAR) subunit genes. Positional clustering is useful for improving computational binding site predictions, with potential application to improving our understanding of mammalian gene expression. In particular, predicted regulatory mechanisms in the mammalian GABAAR subunit gene family may open new avenues of research towards understanding this pharmacologically important neurotransmitter receptor system.
PMCID: PMC1807961  PMID: 17204484

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