Comparing transcriptomic analysis using QuantSeq and RNA-Seq with Salmon quantification and Tophat2 mapping

We have compared a relatively new method for RNA 3’ prime sequencing, QuantSeq with traditional RNA-Seq which captures reads from the entire RNA-transcript. In addition, we have compared read quantification using traditional base-to-base mapping (Tophat2) with the new pseudoalignment method Salmon. We have performed this experiment on peripheral blood mononuclear cells (PBMC) stimulated with Poly (I:C), a viral mimic that induces innate antiviral responses. RNA was extracted from six samples of peripheral blood mononuclear cells (PBMC) three of which had been treated with the viral mimic Poly(I:C). Libraries were prepared from the six samples using both the Illumina TruSeq mRNA kit and the QuantSeq 3' sequencing kit. All libraries were sequenced on the Illumina Nextseq 500. The sequenced reads were mapped using Tophat2 or Salmon. Differential expression between the Controls and Poly(I:C) treated samples was assessed using the voom function and the limma package from Bioconductor. Downstream processing was also performed on a subset of the entire reads.