I am looking for a specific gene in my transcriptome data of a non-model species. I aligned all transcripts that were annotated to this particular gene. Now I want to design primers for one specific transcript in the MSA. What is the best way to look for unique regions in a sequence when comparing it to many other sequences in the MSA?
Should I select a unique region "manually" in my sequence of interest and design primers?
Is there a better/faster way, or a software to use, for selecting unique regions in a large MSA?
You could try
PriFi https://www.hsls.pitt.edu/obrc/index.php?page=URL1133980993
The software is a bit outdated but the description fits your problem. Not sure if their web link is still functional.
I also suggest two more recently developed tools used to create primers for MSA. I advise against manually designing a primer for MSA as you have to account for many variables also discussed below
Article PrimerDesign-M: A multiple-alignment based multiple-primer d...
Article A multiple-alignment based primer design algorithm for genet...
As mentioned before, there are software which can be used to get the primers and even primer blast can be use to get the primer candidates, but in the end you will and have to do manual efforts to get it correct.
Dear Esther Senden
I am afraid you might be interested in using DOMINO. This pipeline is intented to develop markers from NGS reads in non-model organisms where no genome is available. Also, there is new option to identify molecular markers, conserved and variables regions directly from MSA. User provides features such as length, variation etc.
Yes, me and other collaborators developed it.
Please take a look and if you can't figure out how to make it work or something is going on please contact us and we would be happy to help you.
http://www.ub.edu/softevol/domino/
https://github.com/molevol-ub/DOMINO
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