A New Pipeline for the Normalization and Pooling of Metabolomics Data

• Main Authors: Vivian Viallon, Mathilde His, Sabina Rinaldi, Marie Breeur, Audrey Gicquiau, Bertrand Hemon, Kim Overvad, Anne Tjønneland, Agnetha Linn Rostgaard-Hansen, Joseph A. Rothwell, Lucie Lecuyer, Gianluca Severi, Rudolf Kaaks, Theron Johnson, Matthias B. Schulze, Domenico Palli, Claudia Agnoli, Salvatore Panico, Rosario Tumino, Fulvio Ricceri, W. M. Monique Verschuren, Peter Engelfriet, Charlotte Onland-Moret, Roel Vermeulen, Therese Haugdahl Nøst, Ilona Urbarova, Raul Zamora-Ros, Miguel Rodriguez-Barranco, Pilar Amiano, José Maria Huerta, Eva Ardanaz, Olle Melander, Filip Ottoson, Linda Vidman, Matilda Rentoft, Julie A. Schmidt, Ruth C. Travis, Elisabete Weiderpass, Mattias Johansson, Laure Dossus, Mazda Jenab, Marc J. Gunter, Justo Lorenzo Bermejo, Dominique Scherer, Reza M. Salek, Pekka Keski-Rahkonen, Pietro Ferrari
• Format: Article
• Language: English
• Published: MDPI AG 2021-09-01
• Series: Metabolites
• Subjects: cancer epidemiology; normalization; pooling; technical variability; metabolomics; metabolites
• Online Access: https://www.mdpi.com/2218-1989/11/9/631

Pooling metabolomics data across studies is often desirable to increase the statistical power of the analysis. However, this can raise methodological challenges as several preanalytical and analytical factors could introduce differences in measured concentrations and variability between datasets. Specifically, different studies may use variable sample types (e.g., serum versus plasma) collected, treated, and stored according to different protocols, and assayed in different laboratories using different instruments. To address these issues, a new pipeline was developed to normalize and pool metabolomics data through a set of sequential steps: (i) exclusions of the least informative observations and metabolites and removal of outliers; imputation of missing data; (ii) identification of the main sources of variability through principal component partial R-square (PC-PR2) analysis; (iii) application of linear mixed models to remove unwanted variability, including samples’ originating study and batch, and preserve biological variations while accounting for potential differences in the residual variances across studies. This pipeline was applied to targeted metabolomics data acquired using Biocrates AbsoluteIDQ kits in eight case-control studies nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Comprehensive examination of metabolomics measurements indicated that the pipeline improved the comparability of data across the studies. Our pipeline can be adapted to normalize other molecular data, including biomarkers as well as proteomics data, and could be used for pooling molecular datasets, for example in international consortia, to limit biases introduced by inter-study variability. This versatility of the pipeline makes our work of potential interest to molecular epidemiologists.