Removing Batch Effects from Longitudinal Gene Expression - Quantile Normalization Plus ComBat as Best Approach for Microarray Transcriptome Data

PLoS One. 2016 Jun 7;11(6):e0156594. doi: 10.1371/journal.pone.0156594. eCollection 2016.


Technical variation plays an important role in microarray-based gene expression studies, and batch effects explain a large proportion of this noise. It is therefore mandatory to eliminate technical variation while maintaining biological variability. Several strategies have been proposed for the removal of batch effects, although they have not been evaluated in large-scale longitudinal gene expression data. In this study, we aimed at identifying a suitable method for batch effect removal in a large study of microarray-based longitudinal gene expression. Monocytic gene expression was measured in 1092 participants of the Gutenberg Health Study at baseline and 5-year follow up. Replicates of selected samples were measured at both time points to identify technical variability. Deming regression, Passing-Bablok regression, linear mixed models, non-linear models as well as ReplicateRUV and ComBat were applied to eliminate batch effects between replicates. In a second step, quantile normalization prior to batch effect correction was performed for each method. Technical variation between batches was evaluated by principal component analysis. Associations between body mass index and transcriptomes were calculated before and after batch removal. Results from association analyses were compared to evaluate maintenance of biological variability. Quantile normalization, separately performed in each batch, combined with ComBat successfully reduced batch effects and maintained biological variability. ReplicateRUV performed perfectly in the replicate data subset of the study, but failed when applied to all samples. All other methods did not substantially reduce batch effects in the replicate data subset. Quantile normalization plus ComBat appears to be a valuable approach for batch correction in longitudinal gene expression data.

Publication types

  • Observational Study

MeSH terms

  • Adult
  • Aged
  • Female
  • Gene Expression Profiling / methods*
  • Humans
  • Longitudinal Studies
  • Male
  • Middle Aged
  • Monocytes / chemistry*
  • Nonlinear Dynamics
  • Oligonucleotide Array Sequence Analysis / methods*
  • Principal Component Analysis
  • Prospective Studies

Grant support

The Gutenberg Health Study is funded through the government of Rhineland-Palatinate („Stiftung Rheinland-Pfalz für Innovation“, contract AZ 961-386261/733), the research programs “Wissenschafft Zukunft” and “Center for Translational Vascular Biology (CTVB)” of the Johannes Gutenberg-University of Mainz, and its contract with Boehringer Ingelheim and PHILIPS Medical Systems, including an unrestricted grant for the Gutenberg Health Study. PSW, SB, TZ and AZ are PIs of the German Center for Cardiovascular Research (DZHK). The project was further supported by the BMBF (German federal ministry of education and research) grant no.01ZX1408A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.