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:
- 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).
- Split into individual FastA files for each sequence (used pyfaidx v0.5.5.2)
- Design primers on AF177226 (COX1) using Primer3.
- Confirm primer specificity using EMBOSS(v6.6.0) primersearch tool to check all individual sequence files for possible matches.
Jupyter Notebook (GitHub):