Gene set enrichment analysis for analyzing large profiling and screening experiments can reveal unifying biological schemes based on previously accumulated knowledge represented as “gene sets”. Most of the existing implementations use a fixed fold-change or P value cutoff to generate regulated gene lists. However, the threshold selection in most cases is arbitrary, and has a significant effect on the test outcome and interpretation of the experiment. We developed a new gene set enrichment analysis method, ie, FDR-FET, which dynamically optimizes the threshold choice and improves the sensitivity and selectivity of gene set enrichment analysis. The procedure translates experimental results into a series of regulated gene lists at multiple false discovery rate (FDR) cutoffs, and computes the P value of the overrepresentation of a gene set using a Fisher’s exact test (FET) in each of these gene lists. The lowest P value is retained to represent the significance of the gene set. We also implemented improved methods to define a more relevant global reference set for the FET. We demonstrate the validity of the method using a published microarray study of three protease inhibitors of the human immunodeficiency virus and compare the results with those from other popular gene set enrichment analysis algorithms. Our results show that combining FDR with multiple cutoffs allows us to control the error while retaining genes that increase information content. We conclude that FDR-FET can selectively identify significant affected biological processes. Our method can be used for any user-generated gene list in the area of transcriptome, proteome, and other biological and scientific applications.

Summary: Dose–response information is critical to understanding drug effects, yet analytical methods for dose–response assays cannot cope with the dimensionality of large-scale screening data such as the microarray profiling data. To overcome this limitation, we developed and implemented the Sigmoidal Dose Response Search (SDRS) algorithm, a grid search-based method designed to handle large-scale dose–response data. This method not only calculates the pharmacological parameters for every assay, but also provides built-in statistic that enables downstream systematic analyses, such as characterizing dose response at the transcriptome level.

Availability: Bio::SDRS is freely available from CPAN (www.cpan.org).

Contacts: ruiruji@gmail.com; bruc@acm.org

Supplementary Information: Supplementary data is available at Bioinformatics online.

The dose response curve is the gold standard for measuring the effect of a drug treatment, but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns, but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose, calculate classical pharmacological parameters such as the EC50, and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib, nilotinib, dasatinib and PD0325901) across a six-logarithm dose range, using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets, dose-dependent effects on cellular processes were identified using automated pathway analysis, and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover, these methods are automated, robust to non-optimized assays, and could be applied to other sources of quantitative data.

Author Summary

Transcriptional profiling is arguably the most powerful hypothesis-free method for investigating biological effects of drugs—so why do the experiments typically use outmoded single-dose designs? Such single-dose experiments will co-mingle effects that can occur with different potency (e.g., effects on the known target versus effects on additional undesired targets). Single-dose experiments have little comparability to the dose-response bioassays, which are now used throughout the drug discovery processes. One reason for the disparity between experimental approaches is that existing analytical methods for dose-response bioassays can't cope with the dimensionality of microarray data: a typical bioassay is optimized for one response, then used to run a screen against thousands of compounds; whereas transcriptional profiling measures thousands of non-optimized responses to a single compound. Conversely, existing methods for microarray data analysis can identify patterns, but provide no quantitative dose-response information. To overcome these problems, we developed novel algorithms and visualization methods that allow anyone to apply transcriptional profiling as a conventional dose-response assay. The approach provides far more information than limited-dose designs, yet is economical (12 arrays/compound). With this new analytical framework, it is now possible to identify distinct transcriptional responses at distinct regions of the dose range, to link these impacts to biological pathways, and to make realistic connections to drug targets and to other bioassays.