Collective analysis of multiple high-throughput gene expression datasets

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University LondonModern technologies have resulted in the production of numerous high-throughput biological datasets. However, the pace of development of capable computational methods does not cope with the pace of generation of new high-throughput datasets. Amongst the most popular biological high-throughput datasets are gene expression datasets (e.g. microarray datasets). This work targets this aspect by proposing a suite of computational methods which can analyse multiple gene expression datasets collectively. The focal method in this suite is the unification of clustering results from multiple datasets using external specifications (UNCLES). This method applies clustering to multiple heterogeneous datasets which measure the expression of the same set of genes separately and then combines the resulting partitions in accordance to one of two types of external specifications; type A identifies the subsets of genes that are consistently co-expressed in all of the given datasets while type B identifies the subsets of genes that are consistently co-expressed in a subset of datasets while being poorly co-expressed in another subset of datasets. This contributes to the types of questions which can addressed by computational methods because existing clustering, consensus clustering, and biclustering methods are inapplicable to address the aforementioned objectives. Moreover, in order to assist in setting some of the parameters required by UNCLES, the M-N scatter plots technique is proposed. These methods, and less mature versions of them, have been validated and applied to numerous real datasets from the biological contexts of budding yeast, bacteria, human red blood cells, and malaria. While collaborating with biologists, these applications have led to various biological insights. In yeast, the role of the poorly-understood gene CMR1 in the yeast cell-cycle has been further elucidated. Also, a novel subset of poorly understood yeast genes has been discovered with an expression profile consistently negatively correlated with the well-known ribosome biogenesis genes. Bacterial data analysis has identified two clusters of negatively correlated genes. Analysis of data from human red blood cells has produced some hypotheses regarding the regulation of the pathways producing such cells. On the other hand, malarial data analysis is still at a preliminary stage. Taken together, this thesis provides an original integrative suite of computational methods which scrutinise multiple gene expression datasets collectively to address previously unresolved questions, and provides the results and findings of many applications of these methods to real biological datasets from multiple contexts.National Institute for Health Research (NIHR) and the Brunel College of Engineering, Design and Physical Science

Paradigm of tunable clustering using binarization of consensus partition matrices (Bi-CoPaM) for gene discovery

Copyright @ 2013 Abu-Jamous et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Clustering analysis has a growing role in the study of co-expressed genes for gene discovery. Conventional binary and fuzzy clustering do not embrace the biological reality that some genes may be irrelevant for a problem and not be assigned to a cluster, while other genes may participate in several biological functions and should simultaneously belong to multiple clusters. Also, these algorithms cannot generate tight clusters that focus on their cores or wide clusters that overlap and contain all possibly relevant genes. In this paper, a new clustering paradigm is proposed. In this paradigm, all three eventualities of a gene being exclusively assigned to a single cluster, being assigned to multiple clusters, and being not assigned to any cluster are possible. These possibilities are realised through the primary novelty of the introduction of tunable binarization techniques. Results from multiple clustering experiments are aggregated to generate one fuzzy consensus partition matrix (CoPaM), which is then binarized to obtain the final binary partitions. This is referred to as Binarization of Consensus Partition Matrices (Bi-CoPaM). The method has been tested with a set of synthetic datasets and a set of five real yeast cell-cycle datasets. The results demonstrate its validity in generating relevant tight, wide, and complementary clusters that can meet requirements of different gene discovery studies.National Institute for Health Researc

Towards tunable consensus clustering for studying functional brain connectivity during affective processing

In the past decades, neuroimaging of humans has gained a position of status within neuroscience, and data-driven approaches and functional connectivity analyses of functional magnetic resonance imaging (fMRI) data are increasingly favored to depict the complex architecture of human brains. However, the reliability of these findings is jeopardized by too many analysis methods and sometimes too few samples used, which leads to discord among researchers. We propose a tunable consensus clustering paradigm that aims at overcoming the clustering methods selection problem as well as reliability issues in neuroimaging by means of first applying several analysis methods (three in this study) on multiple datasets and then integrating the clustering results. To validate the method, we applied it to a complex fMRI experiment involving affective processing of hundreds of music clips. We found that brain structures related to visual, reward, and auditory processing have intrinsic spatial patterns of coherent neuroactivity during affective processing. The comparisons between the results obtained from our method and those from each individual clustering algorithm demonstrate that our paradigm has notable advantages over traditional single clustering algorithms in being able to evidence robust connectivity patterns even with complex neuroimaging data involving a variety of stimuli and affective evaluations of them. The consensus clustering method is implemented in the R package “UNCLES” available on http://cran.r-project.org/web/packages/UNCLES/index.html

UNCLES: Method for the identification of genes differentially consistently co-expressed in a specific subset of datasets

Background: Collective analysis of the increasingly emerging gene expression datasets are required. The recently proposed binarisation of consensus partition matrices (Bi-CoPaM) method can combine clustering results from multiple datasets to identify the subsets of genes which are consistently co-expressed in all of the provided datasets in a tuneable manner. However, results validation and parameter setting are issues that complicate the design of such methods. Moreover, although it is a common practice to test methods by application to synthetic datasets, the mathematical models used to synthesise such datasets are usually based on approximations which may not always be sufficiently representative of real datasets. Results: Here, we propose an unsupervised method for the unification of clustering results from multiple datasets using external specifications (UNCLES). This method has the ability to identify the subsets of genes consistently co-expressed in a subset of datasets while being poorly co-expressed in another subset of datasets, and to identify the subsets of genes consistently co-expressed in all given datasets. We also propose the M-N scatter plots validation technique and adopt it to set the parameters of UNCLES, such as the number of clusters, automatically. Additionally, we propose an approach for the synthesis of gene expression datasets using real data profiles in a way which combines the ground-truth-knowledge of synthetic data and the realistic expression values of real data, and therefore overcomes the problem of faithfulness of synthetic expression data modelling. By application to those datasets, we validate UNCLES while comparing it with other conventional clustering methods, and of particular relevance, biclustering methods. We further validate UNCLES by application to a set of 14 real genome-wide yeast datasets as it produces focused clusters that conform well to known biological facts. Furthermore, in-silico-based hypotheses regarding the function of a few previously unknown genes in those focused clusters are drawn. Conclusions: The UNCLES method, the M-N scatter plots technique, and the expression data synthesis approach will have wide application for the comprehensive analysis of genomic and other sources of multiple complex biological datasets. Moreover, the derived in-silico-based biological hypotheses represent subjects for future functional studies.The National Institute for Health Research (NIHR) under its Programme Grants for Applied Research Programme (Grant Reference Number RP-PG-0310-1004)

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C or C photosynthesis. As part of a multinational effort to introduce C traits into rice to boost crop yield, candidate regulators of C leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C -like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C plants

In vitro downregulated hypoxia transcriptome is associated with poor prognosis in breast cancer

© The Author(s), 2017. Background Hypoxia is a characteristic of breast tumours indicating poor prognosis. Based on the assumption that those genes which are up-regulated under hypoxia in cell-lines are expected to be predictors of poor prognosis in clinical data, many signatures of poor prognosis were identified. However, it was observed that cell line data do not always concur with clinical data, and therefore conclusions from cell line analysis should be considered with caution. As many transcriptomic cell-line datasets from hypoxia related contexts are available, integrative approaches which investigate these datasets collectively, while not ignoring clinical data, are required. Results We analyse sixteen heterogeneous breast cancer cell-line transcriptomic datasets in hypoxia-related conditions collectively by employing the unique capabilities of the method, UNCLES, which integrates clustering results from multiple datasets and can address questions that cannot be answered by existing methods. This has been demonstrated by comparison with the state-of-the-art iCluster method. From this collection of genome-wide datasets include 15,588 genes, UNCLES identified a relatively high number of genes (>1000 overall) which are consistently co-regulated over all of the datasets, and some of which are still poorly understood and represent new potential HIF targets, such as RSBN1 and KIAA0195. Two main, anti-correlated, clusters were identified; the first is enriched with MYC targets participating in growth and proliferation, while the other is enriched with HIF targets directly participating in the hypoxia response. Surprisingly, in six clinical datasets, some sub-clusters of growth genes are found consistently positively correlated with hypoxia response genes, unlike the observation in cell lines. Moreover, the ability to predict bad prognosis by a combined signature of one sub-cluster of growth genes and one sub-cluster of hypoxia-induced genes appears to be comparable and perhaps greater than that of known hypoxia signatures. Conclusions We present a clustering approach suitable to integrate data from diverse experimental set-ups. Its application to breast cancer cell line datasets reveals new hypoxia-regulated signatures of genes which behave differently when in vitro (cell-line) data is compared with in vivo (clinical) data, and are of a prognostic value comparable or exceeding the state-of-the-art hypoxia signatures.Dr. Abu-Jamous would like to acknowledge the financial assistance from Brunel University London. Professors Buffa and Harris acknowledge support from Cancer Research UK, EU framework 7, and the Oxford NIHR Biomedical Research Centre. Professor Harris acknowledges support from the Breast Cancer Research Foundation. Professor Nandi would like to acknowledge that this work was partly supported by the National Science Foundation of China grant number 61520106006 and the National Science Foundation of Shanghai grant number 16JC1401300. The funding bodies have no role in the design of the study, in the collection, analysis, and interpretation of data, or in writing the manuscript

Clust: automatic extraction of optimal co-expressed gene clusters from gene expression data

Abstract Identifying co-expressed gene clusters can provide evidence for genetic or physical interactions. Thus, co-expression clustering is a routine step in large-scale analyses of gene expression data. We show that commonly used clustering methods produce results that substantially disagree and that do not match the biological expectations of co-expressed gene clusters. We present clust, a method that solves these problems by extracting clusters matching the biological expectations of co-expressed genes and outperforms widely used methods. Additionally, clust can simultaneously cluster multiple datasets, enabling users to leverage the large quantity of public expression data for novel comparative analysis. Clust is available at https://github.com/BaselAbujamous/clust

Clust_100_GE_datasets

<p>100 microarray and RNA-seq gene expression datasets from five model species (human, mouse, fruit fly, arabidopsis plants, and baker's yeast). These datasets represent the benchmark set that was used to test our <em>clust</em> clustering method and to compare it with seven widely used clustering methods (Cross-Clustering, <em>k</em>-means, self-organising maps, MCL, hierarchical clustering, CLICK, and WGCNA). This data resource includes raw data files, pre-processed data files, clustering results, clustering results evaluation, and scripts.</p> <p>The files are split into eight zipped parts, 100Datasets_0.zip to 100Datasets_7.zip. The contents of the three zipped files should be extracted to a single folder (e.g. 100Datasets).</p> <p>Below is a thorough description of the files and folders in this data resource.</p> <p><strong>Scripts</strong></p> <p>The scripts used to apply each one of the clustering methods to each one of the 100 datasets and to evaluate their results are all included in the folder (scripts/).</p> <p><strong>Datasets and clustering results (folders starting with D)</strong></p> <p>The datasets are labelled as D001 to D100. Each dataset has two folders: D###/ and D###_Res/, where ### is the number of the dataset. The first folder only includes the raw dataset while the second folder includes the results of applying the clustering methods to that dataset. The files ending with _B.tsv include clustering results in the form of a partition matrix. The files ending with _E include metrics evaluating the clustering results. The files ending with _go and _go_E respectively include the enriched GO terms in the clustering results and evaluation metrics of these GO terms. The files ending with _REACTOME and _REACTOME_E are similar to the GO term files but for the REACTOME pathway enrichment analysis. Each of these D###_Res/ folders includes a sub-folder "ParamSweepClust" which includes the results of applying <em>clust</em> multiple times to the same dataset while sweeping some parameters.</p> <p><strong>Large datasets analysis results</strong></p> <p>The folder LargeDatasets/  includes data and results for what we refer to as "large" datasets. These are 19 datasets that have more than 50 samples including replicates and have not therefore been included in the set of 100 datasets. However, they fit all of the other dataset selection criteria. We have compared <em>clust</em> with the other clustering methods over these datasets to demonstrate that <em>clust</em> still outperforms other datasets over larger datasets. This folder includes folders LD001/ to LD019/ and LD001_Res/ to LD019_Res/. These have similar format and contents as the D###/ and D###_Res/ folders described above.</p> <p><strong>Simultaneous analysis of multiple datasets (folders starting with MD)</strong></p> <p>As our <em>clust</em> method is design to be able to extract clusters from multiple datasets simultaneously, we also tested it over multiple datasets. All folders starting with MD_ are related to "multiple datasets (MD)" results. Each MD experiment simultaneously analyses <em>d</em> randomly selected datasets either out of a set of 10 arabidopsis datasets or out of a set of 10 yeast datasets. For each one of the two species, all <em>d</em> values from 2 to 10 were tested, and at each one of these <em>d</em> values, 10 different runs were conducted, where at each run a different subset of <em>d</em> datasets is selected randomly.</p> <p>The folders MD_10A and MD_10Y include the full sets of 10 arabidposis or 10 yeast datasets, respectively. Each folder with the format MD_10#_d#_Res## includes the results of applying the eight clustering methods at one of the 10 random runs of one of the selected <em>d</em> values. For example, the "MD_10A_d4_Res03/" folder includes the clustering results of the 3^rd random selection of 4 arabidopsis datasets (the letter A in the folder's name refers to arabidopsis).</p> <p>Our <em>clust</em> method is applied directly over multiple datasets where each dataset is in a separate data file. Each "MD_10#_d#_Res##" folder includes these individual files in a sub-folder named "Processed_Data/". However, the other clustering methods only accept a single input data file. Therefore, the datasets are merged first before being submitted to these methods. Each "MD_10#_d#_Res##" folder includes a file "X_merged.tsv" for the merged data.</p> <p><strong>Evaluation metrics (folders starting with Metrics)</strong></p> <p>Each clustering results folder (D##_Res or  MD_10#_d#_Res##) includes some clustering evaluation files ending with _E. This information is combined into tables for all datasets, and these tables appear in the folders starting with "Metrics_".</p> <p><strong>Other files and folders</strong></p> <p>The GO folder includes the reference GO term annotations for arabidopsis and yeast. Similarly, the REACTOME folder includes the reference REACTOME pathway annotations for arabidopsis and yeast. The Datasets file includes a TAB delimited table describing the 100 datasets. The SearchCriterion file includes the objective methodology of searching the NCBI database to select these 100 datasets. The Specials file includes some special considerations for couple of datasets that differ a bit from what is described in the SearchCriterion file. The Norm### files and the files in the Reps/ folder describe normalisation codes and replicate structures for the datasets and were fed to the <em>clust</em> method as inputs. The Plots/ folder includes plots of the gene expression profiles of the individual genes in the clusters generated by each one of the eight methods over each one of the 100 datasets. Only up to 14 clusters per method are plotted.</p> <p> </p

Integrative cluster analysis in bioinformatics

Clustering techniques are increasingly being put to use in the analysis of high-throughput biological datasets. Novel computational techniques to analyse high throughput data in the form of sequences, gene and protein expressions, pathways, and images are becoming vital for understanding diseases and future drug discovery. This book details the complete pathway of cluster analysis, from the basics of molecular biology to the generation of biological knowledge. The book also presents the latest clustering methods and clustering validation, thereby offering the reader a comprehensive review