pcr.seqs :: Is my 16S primer pair not okay?

Commands in mothur
1

I am trying to obtain Silva coordinates using the 16S V4 Primer pair I was provided.
I end up getting 0-byte outcome for ( silva.full_v123.pcr.fasta )

Procedure followed:

I generated silva.full_v123.fasta, following instructions from README for the SILVA v123 reference files.

When I run pcr.seqs with the company provided 16 V4 Forward and Reverse_Complement( Reverse ) primer,
I end up getting 0-byte outcome. For example:

mothur “#pcr.seqs(inputdir=./data2/, fasta=silva.full_v123.fasta, oligos=pcrTest.oligos, keepdots=F, processors=8)”

Contents of my Oligos file: pcrTest.oligos
primer GACGCTCTTCCGATCTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA ATTAGAWACCCVHGTAGTCCGGCTGACTGACTAGATCGGAAGAGCACA V4_4_16S

Further, I also don’t see this primer pair in Escherichia coli str. K-12 substr. MG1655 .fna file.

I am using mothur/1.39.5. I will be very grateful if you could please advise me what I need to do to fix my problem? Thanks.

2

I wish to clarify that the contents of my oligos file are in one single line.

3

The problem is your primer file. The oligo sequences includes a lot of extra stuff, barcodes etc.
The proper sequences are: F: red R: green
GACGCTCTTCCGATCTTATGGTAATT GTGTGCCAGCMGCCGCGGTAA ATTAGAWACCCVHGTAGTCCGGCTGACTGACTAGATCGGAAGAGCACA
(The green part has to be inverted)

V4f: GTGCCAGCMGCCGCGGTAA

V4r: GGACTACHVGGGTWTCTAAT

4

To user: toneta
I am Super thankful for your most kind timely help.
In fact, I was struggling for quite some time – did various testing – finally, I posted my question here and you solved it !! Thanks again.