The Vacc-SeqQC project: Benchmarking RNA-Seq for clinical vaccine studies

Johannes B. Goll, Steven E. Bosinger, Travis L. Jensen, Hasse Walum, Tyler Grimes, Gregory K. Tharp, Muktha S. Natrajan, Azra Blazevic, Richard D. Head, Casey E. Gelber, Kristen J. Steenbergen, Nirav B. Patel, Patrick Sanz, Nadine G. Rouphael, Evan J. Anderson, Mark J. Mulligan, Daniel F. Hoft

Research output: Contribution to journalArticlepeer-review


Introduction: Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial. Methods: We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths. Results and Discussion: Our results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.

Original languageEnglish
Article number1093242
JournalFrontiers in immunology
StatePublished - Jan 19 2023


  • ERCC
  • RNA-Seq
  • gene filtering
  • read length
  • reproducibility
  • sequencing depth
  • statistical power
  • tularemia vaccine (DVC-LVS)


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