Question: Mothur: following SOP, received a weird alignment, screen.seqs failing
0
gravatar for gwrathe
17 months ago by
gwrathe0
gwrathe0 wrote:

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
ADD COMMENTlink written 17 months ago by gwrathe0

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

ADD REPLYlink written 17 months ago by Asaf8.4k

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.

ADD REPLYlink written 17 months ago by gwrathe0

So what is in Silva.v4.fasta?

ADD REPLYlink written 17 months ago by Asaf8.4k

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.

ADD REPLYlink modified 17 months ago • written 17 months ago by gwrathe0
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