Primer3: p3in failure

Question

[putita.jira]

Dear all

I'm using MISA and Primer3 in pipeline. After I got *.misa file, I put this syntax.

p3_in.pl FASTA.fa.misa

Then I got empty file of *.p3in. Is it normal?

By the way, I still tried to run further syntax.

primer3_core < FASTA.fa.p3in > FASTA.fa.p3out

This one showed me the description and usage of Primer3.

I put further syntax which is...

p3_out.pl FASTA.fa.p3out FASTA.fa.misa

It showed the error message like this.

Use of uninitialized value $count in concatenation (.) or string at C:\Users\admin\Documents\Work\NGS_results\MISA\p3_out.pl line 76.

Primer modelling was successful for sequences. Use of uninitialized value $count_failed in concatenation (.) or string at C:\Users\admin\Documents\Work\NGS_results\MISA\p3_out.pl line 77. Primer modelling failed for sequences.

Could anyone please help me to solve this issue?

Thank you

[Viz]

I would recommend putting your .misa file into http://bioinfo.ut.ee/primer3/ and seeing if things work from there.

Also check: SSR mining and primer design (MISA and Primer3)

Answer 1

[h.mon]

The fasta file has spaces at the headers, and the .misa file has replaced these spaces by underscores - and you already know this: C: SSR mining and primer design (MISA and Primer3) .

You already asked the same here: C: SSR mining and primer design (MISA and Primer3) . My comment there already hinted at the most likely problem (spaces in fasta headers), and yet you didn't include this information in this post. Please be mindful of others efforts and put some more care into your questions, adding the relevant details from the start, and by not posting the same question at multiple places.

[putita.jira]

Sorry h.mon.

At that time, I was afraid that no one would read comment in the previous post and found that I have new problem so I decided to post new question here. Btw, I don't know how to delete this post. Thank you for your effort. I will never post the same question again.

[h.mon]

Sorry, I have been too harsh. The problem is not exactly opening a new question, is opening a new question without providing proper context. You should have:

1. linked the old thread, stating the new question was a development of the older one,

2. already include the information about contig names!

Anyway, did replacing the spaces by underscores at the fasta file solve the problem?