The application of MANOVA to analyse metabolomic data from factorially designed experiments

Metabolomic technologies produce complex multivariate datasets and researchers are faced with the daunting task of extracting information from these data. Principal component analysis (PCA) has been widely applied in the field of metabolomics to reduce data dimensionality and for visualising trends within the complex data. Although PCA is very useful, it cannot handle multi-factorial experimental designs and, often, clear trends of biological interest are not observed when plotting various PC combinations. Even if patterns are observed, PCA provides no measure of their significance. Multivariate analysis of variance (MANOVA) applied to these PCs enables the statistical evaluation of main treatments and, more importantly, their interactions within the experimental design. The power and scope of MANOVA is demonstrated through two different factorially designed metabolomic investigations using ethylene signalling mutants and their wild-type. One investigation has multiple experimental factors including challenge with the economically important pathogen and also replicate experiments, while the second has different sample preparation methods and one level of replication ‘nested’ within the design. In both investigations there are specific factors of biological interest and there are also factors incorporated within the experimental design, which affect the data. The versatility of MANOVA is displayed by using data from two different metabolomic techniques; profiling using direct injection mass spectroscopy (DIMS) and fingerprinting using fourier transform infra-red (FT-IR) spectroscopy. MANOVA found significant main effects and interactions in both experiments, allowing a more complete and comprehensive interpretation of the variation within each investigation, than with PCA alone. Canonical variate analysis (CVA) was applied to investigate these effects and their biological significance. In conclusion, the application of MANOVA followed by CVA provided extra information than PCA alone and proved to be a valuable statistical addition in the overwhelming task of analysing metabolomic data.