RNA-seq differential expression studies: More sequence or more replication?

Abstract

Motivation: RNA-seq is replacing microarrays as the primary tool for gene expression studies. Many RNA-seq studies have used insufficient biological replicates, resulting in low statistical power and inefficient use of sequencing resources.Results: We show the explicit trade-off between more biological replicates and deeper sequencing in increasing power to detect differentially expressed (DE) genes. In the human cell line MCF7, adding more sequencing depth after 10 M reads gives diminishing returns on power to detect DE genes, whereas adding biological replicates improves power significantly regardless of sequencing depth. We also propose a cost-effectiveness metric for guiding the design of large-scale RNA-seq DE studies. Our analysis showed that sequencing less reads and performing more biological replication is an effective strategy to increase power and accuracy in large-scale differential expression RNA-seq studies, and provided new insights into efficient experiment design of RNA-seq studies. © 2013 The Author 2013.