In preparation for designing primers for developing a geoduck vitellogenin qPCR assay, I annotated a de novo geoduck transcriptome assembly last week. Next up, identify vitellogenin genes, design primers, confirm their specificity, and order them!
All of this was done in a Jupyter Notebook on my computer (Swoose).
Jupyter notebook (GitHub):
Annoated transcriptome FastA (271MB):
Although everything is explained pretty well in the Jupyter Notebook, here’s the brief rundown of the process:
- Download FastA file.
- Split into individual FastA files for each sequence (used pyfaidx v0.5.5.2)
- Identify sequences (in original FastA file, not individual files) annotated as vitellogenin.
- Design primers on best vitellogenin match (based on TransDecoder score and BLASTp e-values) using Primer3.
- Confirm primer specificity using EMBOSS(v6.6.0) primersearch tool to check all individual sequence files for possible matches.