MetaboClust: Using interactive time-series cluster analysis to relate metabolomic data with perturbed pathways.

MOTIVATION: Modern analytical techniques such as LC-MS, GC-MS and NMR are increasingly being used to study the underlying dynamics of biological systems by tracking changes in metabolite levels over time. Such techniques are capable of providing information on large numbers of metabolites simultaneously, a feature that is exploited in non-targeted studies. However, since the dynamics of specific metabolites are unlikely to be known a priori this presents an initial subjective challenge as to where the focus of the investigation should be. Whilst a number of feed-forward software tools are available for manipulation of metabolomic data, no tool centralizes on clustering and focus is typically directed by a workflow that is chosen in advance. RESULTS: We present an interactive approach to time-course analyses and a complementary implementation in a software package, MetaboClust. This is presented through the analysis of two LC-MS time-course case studies on plants (Medicago truncatula and Alopecurus myosuroides). We demonstrate a dynamic, user-centric workflow to clustering with intrinsic visual feedback at all stages of analysis. The software is used to apply data correction, generate the time-profiles, perform exploratory statistical analysis and assign tentative metabolite identifications. Clustering is used to group metabolites in an unbiased manner, allowing pathway analysis to score metabolic pathways, based on their overlap with clusters showing interesting trends.

Integrating transcriptomic techniques and k-means clustering in metabolomics to identify markers of abiotic and biotic stress in Medicago truncatula.

INTRODUCTION: Nitrogen-fixing legumes are invaluable crops, but are sensitive to physical and biological stresses. Whilst drought and infection from the soil-borne pathogen Fusarium oxysporum have been studied individually, their combined effects have not been widely investigated. OBJECTIVES: We aimed to determine the effect of combined stress using methods usually associated with transcriptomics to detect metabolic differences between treatment groups that could not be identified by more traditional means, such as principal component analysis and partial least squares discriminant analysis. METHODS: Liquid chromatography-high resolution mass spectrometry data from the root and leaves of model legume Medicago truncatula were analysed using Gaussian Process 2-Sample Test, k-means cluster analysis and temporal clustering by affinity propagation. RESULTS: Metabolic differences were detected: we identified known stress markers, including changes in concentration for sucrose and citric acid, and showed that combined stress can exacerbate the effect of drought. Changes in roots were found to be smaller than those in leaves, but differences due to Fusarium infection were identified. The transfer of sucrose from leaves to roots can be seen in the time series using transcriptomic techniques with the metabolomics time series. Other metabolite concentrations that change as a result of treatment include phosphoric acid, malic acid and tetrahydroxychalcone. CONCLUSIONS: Probing metabolomic data with transcriptomic tools provides new insights and could help to identify resilient plant varieties, thereby increasing future crop yield and improving food security.