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.
Jupyter Notebook (GitHub):
All files were transferred to Gannet using
Primer3 human-readable output (TXT):
Here’s the info on the Primer3 top primer pair (scroll to the right to see primer sequences):
PRIMER PICKING RESULTS FOR AF177226.1_cds_AAF20053.1_12 No mispriming library specified Using 0-based sequence positions OLIGO start len tm gc% any_th 3'_th hairpin seq LEFT PRIMER 205 19 59.54 57.89 0.00 0.00 34.59 GGGGGTTTGGTAACTGGCT RIGHT PRIMER 352 18 59.88 61.11 0.00 0.00 0.00 CCTGCCCCAACTCCGTTT SEQUENCE SIZE: 1518 INCLUDED REGION SIZE: 1518 PRODUCT SIZE: 148, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00
Primer3 Primer Design Parameters (TXT):
primersearch tool produced two matches (TXT):
The first match (nc_001276.primersearch) is a match in the full mt genome, which corresponds to the COX1 coding sequence location.
The second match (nw_011935054.primersearch) is within a scaffold of the full C.gigas genome. This result suggests that the full genome includes mitochondrial sequences, as we would not expect mtDNA sequences to be found in the nuclear DNA.
These results confirm that this primer set is specific for COX1.
Will order this primer set.