Workflow for Evaluating Normalization Tools for Omics Data Using Supervised and Unsupervised Machine Learning

To achieve high quality omics results, systematic variability in mass spectrometry (MS) data must be adequately addressed. Effective data normalization is essential for minimizing this variability. The abundance of approaches and the data-dependent nature of normalization have led some researchers to develop open-source academic software for choosing the best approach. While these tools are certainly beneficial to the community, none of them meet all of the needs of all users, particularly users who want to test new strategies that are not available in these products. Herein, we present a simple and straightforward workflow that facilitates the identification of optimal normalization strategies using straightforward evaluation metrics, employing both supervised and unsupervised machine learning. The workflow offers a "DIY" aspect, where the performance of any normalization strategy can be evaluated for any type of MS data. As a demonstration of its utility, we apply this workflow on two distinct datasets, an ESI-MS dataset of extracted lipids from latent fingerprints and a cancer spheroid dataset of metabolites ionized by MALDI-MSI, for which we identified the best-performing normalization strategies.