Discussion
RNA-Seq offers a large amount of flexibility in the design of experiments, and many technical parameters such as read length, sequencing depth, and replication (i.e. sample size) can be varied to match the needs of an experiment. In addition, choices of the lowly-expressed gene, and fold-change thresholds have an impact on downstream analysis. The goal of most RNA-Seq experiments is to detect genes with statistically significant differences in expression across conditions. Several studies in recent years have sought to investigate how technical parameters influence the composition of DEGs and to provide standards for the design of RNA-Seq experiments. The contributions of technical variation to RNA-Seq analysis have been reduced in recent years due to the improvements in library preparation methods and widespread use of Illumina sequencing; however, the contribution of biological variation remains unique to each dataset.With this in mind, we sought to provide general recommendations for conducting RNA-Seq in clinical vaccine studies using real-world data generated from a vaccine clinical trial. To achieve this, we assessed human blood PBMC transcriptomes for 10 healthy subjects at Days 0 (pre-vaccination), 1, 2, 7, and 14 following vaccinations with a 


