Sam’s Notebook: Primer Design – Geoduck Vitellogenin using Primer3

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):

• 20181128_swoose_geoduck_vitellogenin_analysis.ipynb

Annoated transcriptome FastA (271MB):

• 20181121_geo_transdecoder/20180827_trinity_geoduck.fasta.transdecoder.cds

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

1. Download FastA file.
2. Split into individual FastA files for each sequence (used pyfaidx v0.5.5.2)
3. Identify sequences (in original FastA file, not individual files) annotated as vitellogenin.
4. Design primers on best vitellogenin match (based on TransDecoder score and BLASTp e-values) using Primer3.
5. Confirm primer specificity using EMBOSS(v6.6.0) primersearch tool to check all individual sequence files for possible matches.