RNA-Sequencing analysis methods are rapidly evolving, and the tool choice for each step of one common workflow, differential expression analysis, which includes read alignment, expression modeling, and differentially expressed gene identification, has a dramatic impact on performance characteristics. Although a number of workflows are emerging as high performers that are robust to diverse input types, the relative performance characteristics of these workflows when either read depth or sample number is limited-a common occurrence in real-world practice-remain unexplored.
Here, UCSF researchers evaluate the impact of varying read depth and sample number on the performance of differential gene expression identification workflows, as measured by precision, or the fraction of genes correctly identified as differentially expressed, and by recall, or the fraction of differentially expressed genes identified. They focus their analysis on 30 high-performing workflows, systematically varying the read depth and number of biological replicates of patient monocyte samples provided as input. They find that, in general for most workflows, read depth has little effect on workflow performance when held above two million reads per sample, with reduced workflow performance below this threshold. The greatest impact of decreased sample number is seen below seven samples per group, when more heterogeneity in workflow performance is observed. The choice of differential expression identification tool, in particular, has a large impact on the response to limited inputs.
Sample number’s impact on performance
Precision and recall, averaged over the 10 iterations at a given sample number and read depth, split by read depth (columns) and sample number (rows). Values for each workflow (read aligner, expression modeler, and differential expression tool) are averaged and displayed separately. Points represent mean; bars represent standard deviation; colors represent differential expression tool. Red solid line represents linear regression line for plotted data. R2 value corresponds to plotted data. Gray dashed line represents linear regression fit of the first row of data for each column, superimposed over subsequent rows for comparison
Among the tested workflows, the recall/precision balance remains relatively stable at a range of read depths and sample numbers, although some workflows are more sensitive to input restriction. At ranges typically recommended for biological studies, performance is more greatly impacted by the number of biological replicates than by read depth. Caution should be used when selecting analysis workflows and interpreting results from low sample number experiments, as all workflows exhibit poorer performance at lower sample numbers near typically reported values, with variable impact on recall versus precision. These analyses highlight the performance characteristics of common differential gene expression workflows at varying read depths and sample numbers, and provide empirical guidance in experimental and analytical design.