Primers for a different genomes

[lupaupa1]

Hello, I want to make pcr primers for a several different genomes to make PCR in one pool. Maybe I am a little bit stupid, but I dont understand, is there a way how to do it with your program? Will NGS-PrimerPlex automatically check them for cross dimers?

Thanks a lot.

[borobovav]

Hello, @lupaupa1. I suggest, you can simply merge several genomes in one .fna (be careful that the names of the chromosomes are not repeated).

[somakchowdhury]

Hi @aakechin @borobovav

Thanks for this excellent bioinformatics tool.

I have few viral genomes for which I want to design primers. When I use their genbank files the getgeneregions.py command i get the following error and a blank region file

Reading input file and getting chromosome numbers...
E6
E7
E1
E2
E4
E5
L2
L1
Done
Getting genome regions for the input genes...
E6
WARNING (7)! mRNA feature was not found in the GenBank file ref/PPH16.gb for the gene E6!
ERROR (4)! Exon 82 includes noncoding sequences or does not exist.
In the first case, use parameter -noncoding. In the second one, correct the mistake

My gene list file contents are as follows:

E6 83-559
E7 562-858
E1 865-2813
E2 2755-3852
E4 3332-3619
E5 3863-4099
L2 4235-5656
L1 5559-7154

What am I doing wrong?

Is it possible to simply give the entire genome without regions for primer design as my genomes don't have any introns and exons?

Alternatively can I give gene fasta in one file and get multiplexed primers for the gene set.

Any help in this regard is much appreciated..
Best,
SC

[aakechin]

@somakchowdhury Thank you for the question! In the gene list file the second column should contain exon numbers. You can define only gene numbers, and the program will design primers for all exons.
For the entire genome, you can skip the step of extracting gene coordinates, and define manually all regions that you need in the regions file. Or if you need to amplify whole genome, write chromosome number and start-end of the genome. However, I think you will not be able to set from 1 to the end, so you will have to set start and end as values +30 from the start or -30 from the end.
Also, you can use multi-fasta file with whole gene sequences, and define coordinates of target amplicons in the regions file based on your multi-fasta file. In this case, the program will also sort the primers designed into multiplex reaction(s).

[somakchowdhury]

Thank you @aakechin for the input. I will try this.

[Syed-NMBU]

@lupaupa1 Hello, I also want to design primers for multiplex PCR reaction. Have you successfully used this python code for that purpose? Does it helpful for you? Do you have any email?