How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%-40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more…

• W
  Wellcome

  Awards: WT097945, WT083481, 92530/Z/10/Z, WT092340, 098439/Z/12, 095062
• WT
  Wellcome Trust

  Awards: WT092340, WT083481
• DF
  Directorate for Biological Sciences
• MR
  Medical Research Council

  Awards: MR/K001744/1, MR/K001744/1
• BA
  Biotechnology and Biological Sciences Research Council

  Awards: BB/J00247X/1, BB/H002286/1, BB/M010066/1, BB/M004155/1, BB/M004155, BB/M004155/1, BB/H002286/1