hRUV: Hierarchical approach to removal of unwanted variation for large-scale metabolomics data

Abstract Liquid chromatography-mass spectrometry-based metabolomics studies are increasingly applied to large population cohorts, which run for several weeks or even years in data acquisition. This inevitably introduces unwanted intra- and inter-batch variations over time that can overshadow true biological signals and thus hinder potential biological discoveries. To date, normalisation approaches have struggled to mitigate the variability introduced by technical factors whilst preserving biological variance, especially for protracted acquisitions. Here, we propose a study design framework with an arrangement for embedding biological sample replicates to quantify variance within and between batches and a novel workflow that uses these replicates to remove unwanted variation in a hierarchical (hRUV) manner. We use this design to produce a dataset of more than 1,000 human plasma samples run over an extended period of time. We demonstrate significant improvement of hRUV over existing methods in preserving biological signals whilst removing unwanted variation for large scale metabolomics studies. Our novel tools not only provide a strategy for large scale data normalization, but also provides guidance on the design strategy for large omics studies.