Comments on the analysis of unbalanced microarray data

MOTIVATION Permutation testing is very popular for analyzing microarray data to identify differentially expressed (DE) genes; estimating false discovery rates (FDRs) is a very popular way to address the inherent multiple testing problem. However, combining these approaches may be problematic when sample sizes are unequal. RESULTS With unbalanced data, permutation tests may not be suitable because they do not test the hypothesis of interest. In addition, permutation tests can be biased. Using biased P-values to estimate the FDR can produce unacceptable bias in those estimates. Results also show that the approach of pooling permutation null distributions across genes can produce invalid P-values, since even non-DE genes can have different permutation null distributions. We encourage researchers to use statistics that have been shown to reliably discriminate DE genes, but caution that associated P-values may be either invalid, or a less-effective metric for discriminating DE genes.