How to design primers with primer3_core

Question

[Francois Piumi]

Hi, I want to use "primer3_core" to design PCR primers from a multi-fasta file.

The --help says : $ primer3_core < my_input_file

But output said :

PRIMER_ERROR=thermodynamic approach chosen, but path to thermodynamic parameters not specified

Maybe it has something to do with the "PRIMER_THERMODYNAMIC_PARAMETERS_PATH", but the doc is not clear enough for me. There is also no clear example of a command line.

Thanks

Answer 1

[Pierre Lindenbaum]

you have to add a line like:

PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/your/path/to/primer3_config

the primer3_config directory is shipped with primer3 e.g : https://github.com/primer3-org/primer3/tree/master/src/primer3_config

[Francois Piumi]

Thanks Pierre, do you mean :

$ primer3_core < PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/my/path/to/primer3_config my_input_file

?

The primer3_config directory is not found. However it's existing and containing the following files/directories : dangle.dh interpretations loops.ds stack.ds stackmm.ds tetraloop.ds triloop.ds tstack2.ds tstack_tm_inf.ds dangle.ds loops.dh stack.dh stackmm.dh tetraloop.dh triloop.dh tstack2.dh tstack.dh

[Pierre Lindenbaum]

do you mean :

no, a the top of my_input_file

[Francois Piumi]

sorry, it doesn't work....here is my whole command :

$ PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/usr/local/bioinfo/src/Primer3/current/bin/primer3_config $ primer3_core < Sus_scrofa_IFNG_201_sequence.fa

output: PRIMER_ERROR=thermodynamic approach chosen, but path to thermodynamic parameters not specified

[Pierre Lindenbaum]

the input of primer3 is NOT a fasta file. Please, read the manual.

[Francois Piumi]

I took the same example as in the manual and I still have the same error....

[Pierre Lindenbaum]

show me the first lines of your input file please.

[Francois Piumi]

Ok I got it. The "PRIMER_THERMODYNAMIC_PARAMETERS_PATH=/my/path/to/primer3_config/" (with a trailing slash) should be written at the top of the sequence file.

[Francois Piumi]

Merci beaucoup Pierre !

[Pierre Lindenbaum]

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