Sam’s Notebook: Primer Design – Gigas COX1 using Primer3

We’re attempting a quick & dirty comparison of relative mitochondria counts between diploid and triploid C.gigas, so needed a single-copy mitochondrial gene target for qPCR. Selected cytochrome c oxidase subunit 1 (COX1), based on a quick glance at the NCBI mt genome browser for C.gigas NC_001276.

Although everything is explained pretty well in the Jupyter Notebook linked below, here’s the brief rundown of the process:

  1. Download FastA files for C.gigas genome, C.gigas mt genome, C.gigas mt coding sequences (only way I could figure out how to get individual gene nucleotides).
  2. Split into individual FastA files for each sequence (used pyfaidx v0.5.5.2)
  3. Design primers on AF177226 (COX1) using Primer3.
  4. Confirm primer specificity using EMBOSS(v6.6.0) primersearch tool to check all individual sequence files for possible matches.

Jupyter Notebook (GitHub):

Grace’s Notebook: 2015 Oysterseeddia Update