Surface-enhanced Raman scattering (SERS) is a valuable analytical technique for the analysis of biological samples. However, due to the nature of SERS it is often challenging to exploit the generated data to obtain the desired information when no reporter or label molecules are used. Here, the suitability of random forest based approaches is evaluated using SERS data generated by a simulation framework that is also presented. More specifically, it is demonstrated that important SERS signals can be identified, the relevance of predefined spectral groups can be evaluated, and the relations of different SERS signals can be analyzed. It is shown that for the selection of important SERS signals Boruta and surrogate minimal depth (SMD) and for the analysis of spectral groups the competing method Learner of Functional Enrichment (LeFE) should be applied. In general, this investigation demonstrates that the combination of random forest approaches and SERS data is very promising for sophisticated analysis of complex biological samples.

Surface enhanced Raman scattering (SERS) microspectra give biochemical information from nanoscopic volumes in a heterogeneous biomaterial. With the help of machine learning, molecular structure and interactions can be inferred based on SERS data.

Random forest is a popular machine learning approach for the analysis of high-dimensional data because it is flexible and provides variable importance measures for the selection of relevant features. However, the complex relationships between the features are usually not considered for the selection and thus also neglected for the characterization of the analysed samples. Here we propose two novel approaches that focus on the mutual impact of features in random forests. Mutual forest impact (MFI) is a relation parameter that evaluates the mutual association of the features to the outcome and, hence, goes beyond the analysis of correlation coefficients. Mutual impurity reduction (MIR) is an importance measure that combines this relation parameter with the importance of the individual features. MIR and MFI are implemented together with testing procedures that generate P-values for the selection of related and important features. Applications to one experimental and various simulated datasets and the comparison to other methods for feature selection and relation analysis show that MFI and MIR are very promising to shed light on the complex relationships between features and outcome. In addition, they are not affected by common biases, e.g. that features with many possible splits or high minor allele frequencies are preferred. The approaches are implemented in Version 0.3.3 of the R package RFSurrogates that is available at github.com/AGSeifert/RFSurrogates and the data are available at doi.org/10.25592/uhhfdm.12620.

For the untargeted analysis of the metabolome of biological samples with liquid chromatography–mass spectrometry (LC-MS), high-dimensional data sets containing many different metabolites are obtained. Since the utilization of these complex data is challenging, different machine learning approaches have been developed. Those methods are usually applied as black box classification tools, and detailed information about class differences that result from the complex interplay of the metabolites are not obtained. Here, we demonstrate that this information is accessible by the application of random forest (RF) approaches and especially by surrogate minimal depth (SMD) that is applied to metabolomics data for the first time. We show this by the selection of important features and the evaluation of their mutual impact on the multi-level classification of white asparagus regarding provenance and biological identity. SMD enables the identification of multiple features from the same metabolites and reveals meaningful biological relations, proving its high potential for the comprehensive utilization of high-dimensional metabolomics data.

The importance of animal welfare and the organic production of chicken eggs has increased in the European Union in recent years. Legal regulation for organic husbandry makes the production of organic chicken eggs more expensive compared to conventional husbandry and thus increases the risk of food fraud. Therefore, the aim of this study was to develop a non-targeted lipidomic LC-ESI-IM-qToF-MS method based on 270 egg samples, which achieved a classification accuracy of 96.3%. Subsequently, surrogate minimal depth (SMD) was applied to select important variables identified as carotenoids and lipids based on their MS/MS spectra. The LC-MS results were compared with FT-NIR spectroscopy analysis as a low-resolution screening method and achieved 80.0% accuracy. Here, SMD selected parts of the spectrum which are associated with lipids and proteins. Furthermore, we used SMD for low-level data fusion to analyze relations between the variables of the LC-MS and the FT-NIR spectroscopy datasets. Thereby, lipid-associated bands of the FT-NIR spectrum were related to the identified lipids from the LC-MS analysis, demonstrating that FT-NIR spectroscopy partially provides similar information about the lipidome. In future applications, eggs can therefore be analyzed with FT-NIR spectroscopy to identify conspicuous samples that can subsequently be counter-tested by mass spectrometry.