---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: Primer Design - Geoduck Vitellogenin using Primer3
date: '2018-11-29 14:06'
tags:
  - geoduck
  - Panopea generosa
  - vitellogenin
  - Primer3
  - EMBOSS
  - PrimerSearch
  - Jupyter
categories:
  - 2018
  - Miscellaneous
---
In preparation for designing primers for [developing a geoduck vitellogenin qPCR assay](https://github.com/RobertsLab/resources/issues/491), I [annotated a _de novo_ geoduck transcriptome assembly](https://robertslab.github.io/sams-notebook/posts/2018/2018-11-21-Annotation---Geoduck-Transcritpome-with-TransDecoder/) 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](https://github.com/RobertsLab/code/blob/master/notebooks/sam/20181128_swoose_geoduck_vitellogenin_analysis.ipynb)

Annoated transcriptome FastA (271MB):

- [20181121_geo_transdecoder/20180827_trinity_geoduck.fasta.transdecoder.cds](https://gannet.fish.washington.edu/Atumefaciens/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](https://github.com/mdshw5/pyfaidx))

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](https://emboss.sourceforge.net/download/)(v6.6.0) [primersearch tool](http://emboss.sourceforge.net/apps/cvs/emboss/apps/primersearch.html) to check all individual sequence files for possible matches.

---

# RESULTS
All files were transferred to Gannet using ```rsync```.

Output direoctory:

- [20181129_geoduck_vtg_primers/](https://gannet.fish.washington.edu/Atumefaciens/20181129_geoduck_vtg_primers/)


Primer3 human-readable output:
- [20181129_primer3_primers.txt](https://gannet.fish.washington.edu/Atumefaciens/20181129_geoduck_vtg_primers/20181129_primer3_primers.txt)

Here's the info on the Primer3 top primer pair (scroll to the right to see primer sequences):

---

```
PRIMER PICKING RESULTS FOR TRINITY_DN51983_c0_g1_i8.p1.cds

No mispriming library specified
Using 0-based sequence positions
OLIGO            start  len      tm     gc%  any_th  3'_th hairpin seq
LEFT PRIMER       1347   18   59.89   55.56    9.11   0.13   42.06 TTACGCCACGGCAACTGT
RIGHT PRIMER      1471   18   60.05   61.11   10.11   0.00    0.00 CGCAGTGCCAACAAGCTG
SEQUENCE SIZE: 14484
INCLUDED REGION SIZE: 14484

PRODUCT SIZE: 125, PAIR ANY_TH COMPL: 10.66, PAIR 3'_TH COMPL: 0.00
```
---

Primer3 Primer Design Parameters:

- [20181129_geoduck_vtg_primers/20181129_primer3_params.txt](https://gannet.fish.washington.edu/Atumefaciens/20181129_geoduck_vtg_primers/20181129_primer3_params.txt)

The EMBOSS ```primersearch``` tool produced only two matches:

- [trinity_dn51983_c0_g1_i4.primersearch](https://gannet.fish.washington.edu/Atumefaciens/20181129_geoduck_vtg_primers/trinity_dn51983_c0_g1_i4.primersearch)

- [trinity_dn51983_c0_g1_i8.primersearch](https://gannet.fish.washington.edu/Atumefaciens/20181129_geoduck_vtg_primers/trinity_dn51983_c0_g1_i8.primersearch)

The second file is the original FastA file from which the primers were generated, so that's expected.

The first file is the a different isoform of the same gene.

In any case, I'll go ahead and order this primer set.
