Trouble when executing puppy-primers

[caya2]

Hi!
I have some problems with the puppy-primers command. First I tried with my cds.fna files and it did not work so I tried with the test files on this page. I still have the same error message... :

ValueError: Illegal PRIMER_OPT_TM value: 60.0; Illegal PRIMER_MIN_TM value: 58.0; Illegal PRIMER_MAX_TM value: 63.0; Illegal PRIMER_PAIR_MAX_DIFF_TM value: 2.0; Illegal PRIMER_MIN_GC value: 40.0; Illegal PRIMER_MAX_GC value: 60.0

My code is :
puppy-primers -pr INPUT_primerTarget -i ResultDB.tsv -o ResultPrimers

I use PUPpy with conda.

I tried to change the values that does not work put I have the same error message. I did the update yesterday. Bioinformatics is still new for me so maybe it is an easy fix problem, but I really don't understand what I do wrong.

Thanks for your help !

[hghezzi]

Hi!

Thank you for your patience with my delayed response!

Based on your code, I assume that you were able to run puppy-align successfully, managing to create the output folder (default name "alignOUT"). One thing I notice is that you are providing "INPUT_primerTarget" and "ResultDB.tsv" without a specific relative path, which assumes that the folder and file are in the same directory. You can check this by running ls in your current working directory. However, I would expect the file "ResultDB.tsv" to require the path of the output folder "./alignOUT/ResultDB.tsv". The new code may look something like this:

puppy-primers -pr INPUT_primerTarget -i ./alignOUT/ResultDB.tsv -o ResultPrimers

Please let me know if this is not the problem and we can try to work on a new solution!

TLDR: Make sure your filepaths are correct :)

[LucoDevro]

Hi!

I'm having a similar issue. I downloaded a couple of genomic CDS files from NCBI, added cds to the filenames, and distributed them in a target and a non-target folder. I successfully ran puppy-align, which put its output (ResultDB.tsv) in a separate align subfolder, using this command:

puppy-align -pr target/ -nt non_target/ -o align/

However, when I ran puppy-primers using the command below, it fails with similar illegal value errors as above in this thread.

puppy-primers -pr target/ -i ./align/ResultDB.tsv -o primer/ -ng 1 -np 1 -mips 18 -maps 30 -migc 40 -magc 60 -tmd 5 -s 200-5000

Do these Illegal value errors mean I'm putting the primer specs too stringent, or is it a code issue? As you see, I already used some more relaxed values that still work well from our experience in the lab.

The directory structure I run both commands in looks like this:

ls: align non_target primer target

[caya2]

Hi,
thanks for your answer !
Puppy-align did work.
The files where in the good directory, since I wasn't sure if the path was the problem I moved the ResultDB.tsv document in the folder I was in. So the problem is not the path... When I tried to do groups primer with these same files it worked. But specific primers doesn't...

I don't know if you have an other solution...

Thanks!

[mr2raccoon]

Hey, I ran into a similar problem when running puppy-primers. Did anyone find a solution?

I first ran it with standard settings received a similar error and then relaxed the settings to : -mitm 50.0 -matm 67.0 -magc 65.0 and still received a similar error.

  File "primer3/thermoanalysis.pyx", line 1201, in primer3.thermoanalysis._ThermoAnalysis._set_globals_and_seq_args
  File "primer3/thermoanalysis.pyx", line 1182, in primer3.thermoanalysis._ThermoAnalysis._set_globals_and_seq_args
  File "primer3/thermoanalysis.pyx", line 1437, in primer3.thermoanalysis.pdh_wrap_set_seq_args_globals
ValueError: Illegal PRIMER_OPT_TM value: 60.0; Illegal PRIMER_MIN_TM value: 50.0; Illegal PRIMER_MAX_TM value: 67.0; Illegal PRIMER_PAIR_MAX_DIFF_TM value: 2.0; Illegal PRIMER_MIN_GC value: 40.0; Illegal PRIMER_MAX_GC value: 65.0

puppy-align seemed to have worked. I get UniqueGenesList.tsv with 185 unique genes. (annotation columns says no matching annotation for each). Strangely, the UniqueGenesPlot.pdf shows unique Genes = 0 ?
FYI: My target was 1 strain of a species. The non-targets were the cds.fna-files of all other strains of this specific species. Maybe to little variability?

Thanks for creating the tool and any help!

[Hiumin]

I don't know if this information will be helpful to anyone, but puppy-primers was running fine for me until I accidentally used a different micromamba instance (on my university server) to activate the environment. That was when I got this exact same error. Trying out different values for the various "illegal" parameters did not help. The problem persisted with and without non-target CDSs. In the end, everything was fine again after I switched back to the usual micromamba version, but I still don't know why it happened.