Mothur: following SOP, received a weird alignment, screen.seqs failing

Question

[gwrathe]

Hello,

I've been running through the Mothur SOP almost character for character with the V1-V3 region 27f/519r, start = 1046, end = 13127 primers.

I hit the following error message when I ran the 'screen.seqs' command after my alignment.

Running command: remove.seqs(accnos=stability.trim.contigs.good.unique.bad.accnos.temp, count=stability.trim.contigs.good.count_table)
Your file contains only sequences from the .accnos file
Segmentation fault

I went back and looked at the summary files and noticed they seem a bit off compared to the summaries found in the SOP.

Below are the few commands and outputs which came before. I can't figure it out but I'm just a novice, do you guys have any ideas as to why or what I could do differently? Thank you in advance!

mothur > summary.seqs(fasta=silva.v4.fasta)

Using 16 processors.

        Start   End NBases  Ambigs  Polymer NumSeqs
Minimum:    1   7360    337 0   3   1
2.5%-tile:  1   12081   430 0   4   5328
25%-tile:   2   12081   462 0   5   53280
Median:     2   12081   483 0   5   106560
75%-tile:   2   12081   493 0   5   159840
97.5%-tile: 2   12081   545 1   7   207792
Maximum:    74  12081   1596    5   16  213119
Mean:   1   12080   479 0   4
# of Seqs:  213119

It took 4 secs to summarize 213119 sequences.

Output File Names:
/mothur/cyanos/silva.v4.summary


mothur > align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.v4.fasta)

Using 16 processors.

Reading in the /mothur/cyanos/silva.v4.fasta template sequences...  DONE.
It took 48 to read  213119 sequences.

Aligning sequences from /mothur/cyanos/stability.trim.contigs.good.unique.fasta ...
It took 3538 secs to align 240508 sequences.

[WARNING]: 789 of your sequences generated alignments that eliminated too many bases, a list is provided in /mothur/cyanos/stability.trim.contigs.good.unique.flip.accnos.
[NOTE]: 399 of your sequences were reversed to produce a better alignment.

It took 3538 seconds to align 240508 sequences.

Output File Names: 
/mothur/cyanos/stability.trim.contigs.good.unique.align
/mothur/cyanos/stability.trim.contigs.good.unique.align.report
/mothur/cyanos/stability.trim.contigs.good.unique.flip.accnos


mothur > summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table)

Using 16 processors.

        Start   End NBases  Ambigs  Polymer NumSeqs
Minimum:    0   0   0   0   1   1
2.5%-tile:  2   12081   432 0   4   32420
25%-tile:   2   12081   432 0   5   324193
Median:     2   12081   449 0   5   648386
75%-tile:   2   12081   482 0   6   972579
97.5%-tile: 2   12081   486 0   6   1264352
Maximum:    12081   12081   506 0   76  1296771
Mean:   7   12017   454 0   5
# of unique seqs:   240508
total # of seqs:    1296771

It took 8 secs to summarize 1296771 sequences.

Output File Names:
/mothur/cyanos/stability.trim.contigs.good.unique.summary

[Asaf]

Are you using a V4 file you downloaded or did you generate it to your region (v1-v3)?

[gwrathe]

I downloaded the silva.nr_v132.align file and used pcr.seqs with my primer start and end points. I used the output as the align.seqs reference file.

[Asaf]

So what is in Silva.v4.fasta?

[gwrathe]

Renamed output of pcr.seqs. [mothur > rename.file(input=silva.nr_v132.pcr.align, new=silva.v4.fasta)]. I realise the '4' is for the primer region used in the SOP and I just didn't change it, should just be a name and have no effect on the file contents. Sorry for being unclear earlier.