Sam’s Notebook: Primer Design – C.bairdi Primers for Checking RNA for Residual gDNA

Getting ready to run some qPCRs and first we need to confirm that our RNA is actually DNA-free. Before we can do that, we need some primers to use, so I decided to semi-arbitrarily select three different gene targets from our MEGAN6 taxonomic-specific Trinity assembly from 20200122.

I used our recent differential gene expression analysis to identify those genes which were highly differentially expressed in infected vs. uninfected samples.

Overall, the process went something like this:

  1. Sort upregulated genes in infected group by logFC (fold change) to find Trinity transcript IDs of highly expressed genes:
awk 'NR>1' salmon.gene.counts.matrix.infected_vs_uninfected.edgeR.DE_results.P0.05_C1.infected-UP.subset \ | sort -n -k4,4 
  1. Search for some of the highly expressed Trinity IDs in the Trinotate annotations to find SwissProt IDs:
grep "TRINITY_DN6549_c0_g1" \ 20200126.cbai.trinotate_annotation_report.txt 
  1. Copy SwissProt ID (if available) and see what it is on the UniProtKB website.
  2. If interesting (somewhat), search Trinity de novo assembly for transcript sequence.
  3. Use sequence to generate primers on the Primer3 website.