Journal
BMC BIOINFORMATICS
Volume 21, Issue 1, Pages -Publisher
BMC
DOI: 10.1186/s12859-020-03559-6
Keywords
DNA methylation; Simulation; EPIC array; 450 K array; Illumina; Batch effects; ComBat
Categories
Funding
- Charite-Universitatsmedizin Berlin
- Berlin Institute of Health
- German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) [FOR 1617, FR3572/1-1, WA 1539/7-1, HE2597/14-1, HE2597/14/-2]
- German Federal Ministry of Education and Research (Bundesministerium fur Bildung und Forschung) [BMBF 01EE1406G]
Ask authors/readers for more resources
Background Systematic technical effects-also called batch effects-are a considerable challenge when analyzing DNA methylation (DNAm) microarray data, because they can lead to false results when confounded with the variable of interest. Methods to correct these batch effects are error-prone, as previous findings have shown. Results Here, we demonstrate how using the R function ComBat to correct simulated Infinium HumanMethylation450 BeadChip (450 K) and Infinium MethylationEPIC BeadChip Kit (EPIC) DNAm data can lead to a large number of false positive results under certain conditions. We further provide a detailed assessment of the consequences for the highly relevant problem ofp-value inflation with subsequent false positive findings after application of the frequently used ComBat method. Using ComBat to correct for batch effects in randomly generated samples produced alarming numbers of false discovery rate (FDR) and Bonferroni-corrected (BF) false positive results in unbalanced as well as in balanced sample distributions in terms of the relation between the outcome of interest variable and the technical position of the sample during the probe measurement. Both sample size and number of batch factors (e.g. number ofchips) were systematically simulated to assess the probability of false positive findings. The effect of sample size was simulated usingn = 48 up ton = 768 randomly generated samples. Increasing the number of corrected factors led to an exponential increase in the number of false positive signals. Increasing the number of samples reduced, but did not completely prevent, this effect. Conclusions Using the approach described, we demonstrate, that using ComBat for batch correction in DNAm data can lead to false positive results under certain conditions and sample distributions. Our results are thus contrary to previous publications, considering a balanced sample distribution as unproblematic when using ComBat. We do not claim completeness in terms of reporting all technical conditions and possible solutions of the occurring problems as we approach the problem from a clinician's perspective and not from that of a computer scientist. With our approach of simulating data, we provide readers with a simple method to assess the probability of false positive findings in DNAm microarray data analysis pipelines.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available