search

treangenlab / Olivar

Olivar: towards automated variant aware primer design for multiplex tiled amplicon sequencing of pathogens
https://doi.org/10.1038/s41467-024-49957-9
GNU General Public License v3.0
19 stars 2 forks source link

Ambiguous bases in primer design? #7

Open ammaraziz opened 6 months ago

ammaraziz commented 6 months ago

Hi @treangen

Olivar is very interesting in it's approach to primer design. I'm testing it out for a few different organisms. I noticed that while reference can not contain an ambiguous bases, the variation file can have ambiguous bases. However, even if they are included in the variation file, it seems Olivar ignore them in the primers. Interestingly, the graphical output does include the SNP.

It would be super helpful if when Olivar decides on a primer in a particular location where an ambig base exists, the primer would also contain such a base.

Thanks!

Ammar

mxwang66 commented 6 months ago

Hi @ammaraziz,

Thanks for your comment! Ambiguous bases will make energy calculation and primer dimer optimization more difficult, so they are not supported right now. For the variation file, currently only column START, STOP and FREQ is used. In a future update we will include column REF and ALT into the output graphics if provided.

For now, you may replace a ambiguous base with a regular base as input. If a primer overlaps with that base, the ambiguous base can be placed back.

Thank you, Michael

ammaraziz commented 6 months ago

Thanks Michael. Do you think an option to include the ambig base if it overlaps with a primer is possible (with no calculations done on the ambiguous base to simplify things)?

This would simplify things for me. Of course the caveat is that the ambig bases aren't factored into the design step.

mxwang66 commented 6 months ago

Yeah what we can do is converting the ambig base to a regular base (e.g., W to A), run the design and put it back if it sits on a primer. Addtional risk can also be added to that position so the ambig base could be avoided if possible.

ammaraziz commented 6 months ago

Awesome thanks Michael, that'd be a fantastic option to have.

Looking at developing of a few amplicon schemes with Olivar. Looking out for the publication to cite and share around :)