Feature-based molecular networking (FBMN) is a popular analysis approach for liquid chromatography-tandem mass spectrometry-based non-targeted metabolomics data. While processing liquid chromatography-tandem mass spectrometry data through FBMN is fairly streamlined, downstream data handling and statistical interrogation are often a key bottleneck. Especially users new to statistical analysis struggle to effectively handle and analyze complex data matrices. Here we provide a comprehensive guide for the statistical analysis of FBMN results, focusing on the downstream analysis of the FBMN output table. We explain the data structure and principles of data cleanup and normalization, as well as uni- and multivariate statistical analysis of FBMN results. We provide explanations and code in two scripting languages (R and Python) as well as the QIIME2 framework for all protocol steps, from data clean-up to statistical analysis. All code is shared in the form of Jupyter Notebooks ( https://github.com/Functional-Metabolomics-Lab/FBMN-STATS ). Additionally, the protocol is accompanied by a web application with a graphical user interface ( https://fbmn-statsguide.gnps2.org/ ) to lower the barrier of entry for new users and for educational purposes. Finally, we also show users how to integrate their statistical results into the molecular network using the Cytoscape visualization tool. Throughout the protocol, we use a previously published environmental metabolomics dataset for demonstration purposes. Together, the protocol, code and web application provide a complete guide and toolbox for FBMN data integration, cleanup and advanced statistical analysis, enabling new users to uncover molecular insights from their non-targeted metabolomics data. Our protocol is tailored for the seamless analysis of FBMN results from Global Natural Products Social Molecular Networking and can be easily adapted to other mass spectrometry feature detection, annotation and networking tools.

Applied Bioinformatics Department of Computer Science University of Tübingen Tübingen Germany

Bigelow Laboratory for Ocean Sciences East Boothbay ME USA

Bioinformatics Group Wageningen University and Research Wageningen the Netherlands

Boyce Thompson Institute and Department of Chemistry and Chemical Biology Cornell University Ithaca NY USA

Collaborative Mass Spectrometry Innovation Center Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego San Diego CA USA

Department of Analytical Chemistry University of Vienna Vienna Austria

Department of Biochemistry and Microbiology Rhodes University Makhanda South Africa

Department of Biochemistry University of California Riverside Riverside CA USA

Department of Biochemistry University of Johannesburg Johannesburg South Africa

Department of Bioinformatics University of Jena Jena Germany

Department of Biotechnology Engineering School of Lorena University of São Paulo Lorena São Paulo Brazil

Department of Chemistry and Biochemistry University of Denver Denver CO USA

Department of Computer Science University of California Riverside Riverside CA USA

Department of Ecology Behavior and Evolution University of California San Diego San Diego CA USA

Department of Environmental Science Aarhus University Roskilde Denmark

Department of Environmental Systems Analysis University of Tübingen Tübingen Germany

Department of Food Chemistry and Toxicology University of Vienna Vienna Austria

Department of Nutrition Exercise and Sports University of Copenhagen Frederiksberg C Denmark

German Center for Infection Research Partner Site Braunschweig Hannover Braunschweig Germany

Helmholtz Institute for Pharmaceutical Research Saarland Helmholtz Centre for Infection Research Saarbrücken Germany

Institute of Inorganic and Analytical Chemistry University of Münster Münster Germany

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

Laboratorio de Microbiología Molecular y Biotecnología Ambiental Centro de Biotecnología DAL Universidad Técnica Federico Santa María Valparaíso Chile

Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures Braunschweig Germany

Leibniz Institute of Freshwater Ecology and Inland Fisheries Berlin Germany

National Center for Genetic Engineering and Biotechnology National Science and Technology Development Agency Thailand Science Park Pathum Thani Thailand

Saarland University Saarbrücken Germany

School of Marine Sciences Darling Marine Center University of Maine Walpole ME USA

Section for Clinical Mass Spectrometry Danish Center for Neonatal Screening Department of Congenital Disorders Statens Serum Institut Copenhagen S Denmark

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego San Diego CA USA

The Novo Nordisk Foundation for Biosustainability Technical University of Denmark Kongens Lyngby Denmark

Universidad EAFIT Medellín Antioquia Colombia

University of Tübingen Interfaculty Institute of Microbiology and Infection Medicine Tübingen Germany

Virtual Multi Omics Laboratory The Internet Riverside CA USA

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