Single-cell RNA-Sequencing (scRNA-Seq), an advanced sequencing technique, enables biomedical researchers to characterize cell-specific gene expression profiles. Although studies have adapted machine learning algorithms to cluster different cell populations for scRNA-Seq data, few existing methods have utilized machine learning techniques to investigate functional pathways in classifying heterogeneous cell populations. As genes often work interactively at the pathway level, studying the cellular heterogeneity based on pathways can facilitate the interpretation of biological functions of different cell populations. In this paper, we propose a pathway-based analytic framework using Random Forests (RF) to identify discriminative functional pathways related to cellular heterogeneity as well as to cluster cell populations for scRNA-Seq data. We further propose a novel method to construct gene-gene interactions (GGIs) networks using RF that illustrates important GGIs in differentiating cell populations. The co-occurrence of genes in different discriminative pathways and 'cross-talk' genes connecting those pathways are also illustrated in our networks. Our novel pathway-based framework clusters cell populations, prioritizes important pathways, highlights GGIs and pivotal genes bridging cross-talked pathways, and groups co-functional genes in networks. These features allow biomedical researchers to better understand the functional heterogeneity of different cell populations and to pinpoint important genes driving heterogeneous cellular functions.

BACKGROUND: High-throughput sequencing data are widely collected and analyzed in the study of complex diseases in quest of improving human health. Well-studied algorithms mostly deal with single data source, and cannot fully utilize the potential of these multi-omics data sources. In order to provide a holistic understanding of human health and diseases, it is necessary to integrate multiple data sources. Several algorithms have been proposed so far, however, a comprehensive comparison of data integration algorithms for classification of binary traits is currently lacking. RESULTS: In this paper, we focus on two common classes of integration algorithms, graph-based that depict relationships with subjects denoted by nodes and relationships denoted by edges, and kernel-based that can generate a classifier in feature space. Our paper provides a comprehensive comparison of their performance in terms of various measurements of classification accuracy and computation time. Seven different integration algorithms, including graph-based semi-supervised learning, graph sharpening integration, composite association network, Bayesian network, semi-definite programming-support vector machine (SDP-SVM), relevance vector machine (RVM) and Ada-boost relevance vector machine are compared and evaluated with hypertension and two cancer data sets in our study. In general, kernel-based algorithms create more complex models and require longer computation time, but they tend to perform better than graph-based algorithms. The performance of graph-based algorithms has the advantage of being faster computationally. CONCLUSIONS: The empirical results demonstrate that composite association network, relevance vector machine, and Ada-boost RVM are the better performers. We provide recommendations on how to choose an appropriate algorithm for integrating data from multiple sources.

Although many feature selection methods for classification have been developed, there is a need to identify genes in high-dimensional data with censored survival outcomes. Traditional methods for gene selection in classification problems have several drawbacks. First, the majority of the gene selection approaches for classification are single-gene based. Second, many of the gene selection procedures are not embedded within the algorithm itself. The technique of random forests has been found to perform well in high-dimensional data settings with survival outcomes. It also has an embedded feature to identify variables of importance. Therefore, it is an ideal candidate for gene selection in high-dimensional data with survival outcomes. In this paper, we develop a novel method based on the random forests to identify a set of prognostic genes. We compare our method with several machine learning methods and various node split criteria using several real data sets. Our method performed well in both simulations and real data analysis.Additionally, we have shown the advantages of our approach over single-gene-based approaches. Our method incorporates multivariate correlations in microarray data for survival outcomes. The described method allows us to better utilize the information available from microarray data with survival outcomes.

MOTIVATION: There is great interest in pathway-based methods for genomics data analysis in the research community. Although machine learning methods, such as random forests, have been developed to correlate survival outcomes with a set of genes, no study has assessed the abilities of these methods in incorporating pathway information for analyzing microarray data. In general, genes that are identified without incorporating biological knowledge are more difficult to interpret. Correlating pathway-based gene expression with survival outcomes may lead to biologically more meaningful prognosis biomarkers. Thus, a comprehensive study on how these methods perform in a pathway-based setting is warranted. RESULTS: In this article, we describe a pathway-based method using random forests to correlate gene expression data with survival outcomes and introduce a novel bivariate node-splitting random survival forests. The proposed method allows researchers to identify important pathways for predicting patient prognosis and time to disease progression, and discover important genes within those pathways. We compared different implementations of random forests with different split criteria and found that bivariate node-splitting random survival forests with log-rank test is among the best. We also performed simulation studies that showed random forests outperforms several other machine learning algorithms and has comparable results with a newly developed component-wise Cox boosting model. Thus, pathway-based survival analysis using machine learning tools represents a promising approach in dissecting pathways and for generating new biological hypothesis from microarray studies. AVAILABILITY: R package Pwayrfsurvival is available from URL: http://www.duke.edu/~hp44/pwayrfsurvival.htm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: Recent years have seen the development of various pathway-based methods for the analysis of microarray gene expression data. These approaches have the potential to bring biological insights into microarray studies. A variety of methods have been proposed to construct networks using gene expression data. Because individual pathways do not act in isolation, it is important to understand how different pathways coordinate to perform cellular functions. However, there are no published methods describing how to build pathway clusters that are closely related to traits of interest. RESULTS: We propose to build pathway clusters from pathway-based classification methods. The proposed methods allow researchers to identify clusters of pathways sharing similar functions. These pathways may or may not share genes. As an illustration, our approach is applied to three human breast cancer microarray data sets. We found that our methods yielded consistent and interpretable results for these three data sets. We further investigated one of the pathway clusters found using PubMatrix. We found that informative genes in the pathway clusters do have more publications with keywords, like estrogen receptor, compared with informative genes in other top pathways. In addition, using the shortest path analysis in GeneGo's MetaCore and Human Protein Reference Database, we were able to identify the links which connect the pathways without shared genes within the pathway cluster. CONCLUSION: Our proposed pathway clustering methods allow bioinformaticians and biologists to investigate how informative genes within pathways are related to each other and understand possible crosstalk between pathways in a cluster. Therefore, building pathway clusters may lead to a better understanding of molecular mechanisms affecting a trait of interest, and help generate further biological hypotheses from gene expression data.

MOTIVATION: Although numerous methods have been developed to better capture biological information from microarray data, commonly used single gene-based methods neglect interactions among genes and leave room for other novel approaches. For example, most classification and regression methods for microarray data are based on the whole set of genes and have not made use of pathway information. Pathway-based analysis in microarray studies may lead to more informative and relevant knowledge for biological researchers. RESULTS: In this paper, we describe a pathway-based classification and regression method using Random Forests to analyze gene expression data. The proposed methods allow researchers to rank important pathways from externally available databases, discover important genes, find pathway-based outlying cases and make full use of a continuous outcome variable in the regression setting. We also compared Random Forests with other machine learning methods using several datasets and found that Random Forests classification error rates were either the lowest or the second-lowest. By combining pathway information and novel statistical methods, this procedure represents a promising computational strategy in dissecting pathways and can provide biological insight into the study of microarray data. AVAILABILITY: Source code written in R is available from http://bioinformatics.med.yale.edu/pathway-analysis/rf.htm.