Any command line tools that can design primers from a multiple sequence alignment?

Question

[O.rka]

I'm looking for a(n open source) tool that I can use via the command line (preferably installable with conda or pypi) that can take in either 1) fasta targets; or 2) multiple sequence alignment to generate N primers of different confidence levels.

I don't need a forward and reverse pair but mostly a single forward or reverse primer based around 25-30 nt long (possibly using IUPAC characters but not mandatory).

For more context, I've downloaded the 18S fungal sequences and 28S fungal sequences from NCBI. I'm trying to create a forward primer for the 18S and a reverse primer from the 28S sequences so I can amplify out the entire rRNA region including ITS.

All the tools I've seen are either proprietary or web interfaces.

Answer 1

[barslmn]

Primer3 is open source and there is a conda package for it. https://anaconda.org/bioconda/primer3.

You can use something like https://github.com/frba/fasta2primer3 to preprocess your files.

[O.rka]

Thanks, I'll look into this. It doesn't look very user friendly from the manual but I'll give it a try: https://primer3.org/manual.html#inputOutputConventions So I need to convert all of my sequences to a Boulder.IO format to use?

[Ming Tommy Tang]

I wrote a helper function to convert to the Boulder.IO format https://github.com/crazyhottommy/primer3_scATAC_peaks/blob/master/prepare_primer3_input.py

[O.rka]

Is it possible to generate only forward primers based on ~3000 sequences?

[Ming Tommy Tang]

it will give you forward and reverse primers, you can just take the forward one.

[O.rka]

Can I give it the sequences I want it to target?