Pcr.seqs [ERROR]: seqs are not aligned. When using start and end your sequences must be aligned

Commands in mothur
1

I am using Mothur version 1.46.1, within Ubuntu terminal for Windows10
I ran align.seqs with the silva138 reference alignment and the rrnDB fasta file

align.seqs(fasta=data/raw/rrnDB-5.7_16S_rRNA.fasta, reference=data/references/silva_seed/silva.seed_v138.align, flip=T)

This ran as expected, however pcr.seqs on the resulting .align file gave the error:

pcr.seqs(fasta=data/raw/rrnDB-5.7_16S_rRNA.align, start=1044, end=43116, outputdir=data/v19/)

[ERROR]: seqs are not aligned. When using start and end your sequences must be aligned.

I re-downloaded all the files and tried again with the same result

2

Hi ADL,

Can you post the output of running…

summary.seqs(fasta=data/raw/rrnDB-5.7_16S_rRNA.align)

Thanks,
Pat

3

Could you send the rrnDB-5.7_16S_rRNA.fasta file to mothur.bugs@gmail.com so I can take a closer look for you?

4

Have you tried running mothur natively on your Windows machine, https://github.com/mothur/mothur/releases/download/v1.46.1/Mothur.win.zip?

5

mothur > summary.seqs(fasta=data/raw/rrnDB-5.7_16S_rRNA.align)

Using 8 processors.

                Start   End     NBases  Ambigs  Polymer NumSeqs
Minimum:        1044    16429   569     0       4       1
2.5%-tile:      1044    43116   1401    0       5       230
25%-tile:       1044    43116   1437    0       5       2291
Median:         1044    43116   1464    0       6       4582
75%-tile:       1044    43116   1464    0       6       6873
97.5%-tile:     1044    43116   1475    0       7       8934
Maximum:        7922    43116   1576    4       9       9163
Mean:   1048    43111   1453    0       5
# of Seqs:      9163

It took 14 secs to summarize 9163 sequences.

Output File Names:
data/raw/rrnDB-5.7_16S_rRNA.summary
6

Sorry, the formatting is bad. But they sure look aligned

7

Ok, I’m running everything again with the most recent rrnDB files (5.7) and mothur version and now it appears to be working. I did nothing different. Every time before, the command for pcr.seqs terminated after a few seconds, with the error that the .align was not aligned. So I suppose my issue is resolved, but I’m not sure why.

8

Thanks for the update. Just to clarify, is it working using mothur’s windows version or are you still using the Ubuntu terminal on Windows?

9

It’s working on the Ubuntu terminal

10

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