[EMBOSS] eprimer3 troubles

Peter Rice ricepeterm at yahoo.co.uk
Tue Apr 30 08:38:26 UTC 2013

Hi Mark,

On 29/04/2013 20:51, Mark Budde wrote:
> Hi,
> I have spent too many hours trying to get eprimer3 to work on my Mac, and
> am out of ideas.

Easily fixed ... and you've come to the right place to ask.

However, one proviso ... EMBOSS eprimer32 works with primer3 version 
2.2.3 We will support the primer3 2.3.x latest version with the next 
EMBOSS release.

Providing wrappers to external programs becomes difficult when they 
change their options. We were waiting for the primer3 2.3 series to 
stabilise before deciding whether to update eprimer32 or to add a new 
eprimer323 (which may be what we will need to do to support the 2.3.x 
series, with yet another new name for the binary)

> I installed primer3 2.3.5 on Mac. I can run it successfulyl with command
> line.
> When I try and run eprimer3 I get errors.
> $ eprimer32 -sequence=AAAA
> Pick PCR primers and hybridization oligos
> Died: eprimer32 uses external program 'primer32_core' which is not in the
> PATH or defined as EMBOSS_PRIMER32_CORE
>       Part of the 'primer3' package, version 2.2.3, available from the
>       Whitehead Institute. See: http://primer3.sourceforge.net/ The
>       primer3_core application must be renamed to primer32_core
> ...
> This suggested to me that emboss expects primer32_core to be named
> primer32_core, not primer3_core as indicated above.

You misread the instructions. It should indeed be renamed (or copied) 
from the primer3_core that is built when you install primer3 to 
primer32_core (so EMBOSS knows it is running primer 3.2)

> renaming the program to primer32_core, or typing eprimer3 into the command
> line produce the same error:
> ...
> $ eprimer3 -sequence=AAAA
> Pick PCR primers and hybridization oligos
> Error: Failed to open filename 'AAAA'
> Error: Unable to read sequence 'AAAA'
> Died: eprimer3 terminated: Bad value for '-sequence' and no prompt
> ....
> I get this error regardless of what I put as the sequence, or even if I
> input a file.

The message is correct. AAAA is taken as the name of a file. You can 
also use a database name and identifier such as embl:x13776.

However, EMBOSS does also accept the sequence (if it is not too long for 
your system or shell's command line) with the prefix asis:: so you can use

$ eprimer3 -sequence=asis::AAAA

or simply

$ eprimer3 asis::AAAA

You should also give an output file name as such a sequence has no name. 
You can name an "asis" sequence with -sid=myseq on the command line.

> Does anyone have suggestions or potential diagnostics?

For diagnostics, any EMBOSS program can be run with-debug on the command 
line. This produces a file programname.dbg with a trace of messages 
written as it runs.

In this case I am fairly confident we know the problem.


Peter Rice

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