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):
RESULTS
All files were transferred to Gannet using rsync.
Output directory:
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):
The EMBOSS 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.