Errors at screen.seqs after alignment

Commands in mothur
1

I am using mothur 1.48.0 on a unix server, managed by slurm to analyze 12S rRNA, however all sequences are dumped when screened after being aligned to a custom reference and not sure why. I’ve run a very similar script in the past without issue so any help would be greatly appreciated.
The script and outputs from the logfile are below.

mothur > make.contigs(file=tele02.files, deltaq=10, insert=30, trimoverlap=T, processors=30)

mothur > summary.seqs(fasta=current)

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 1 1 0 1 1
2.5%-tile: 1 37 37 0 3 187135
25%-tile: 1 203 203 0 4 1871343
Median: 1 205 205 1 6 3742686
75%-tile: 1 205 205 5 6 5614028
97.5%-tile: 1 210 210 22 7 7298236
Maximum: 1 231 231 94 35 7485370
Mean: 1 197 197 3 5
# of Seqs: 7485370

It took 53 secs to summarize 7485370 sequences.


mothur > screen.seqs(fasta=current, minlength=160, maxambig=0)
It took 17 secs to screen 7485370 sequences, removed 5054917.

mothur > summary.seqs(fasta=current)

Using 30 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 160 160 0 3 1
2.5%-tile: 1 200 200 0 4 60762
25%-tile: 1 203 203 0 5 607614
Median: 1 205 205 0 6 1215227
75%-tile: 1 205 205 0 6 1822840
97.5%-tile: 1 210 210 0 6 2369692
Maximum: 1 230 230 0 16 2430453
Mean: 1 204 204 0 5
# of Seqs: 2430453

It took 17 secs to summarize 2430453 sequences.

mothur > screen.seqs(fasta=current, maxhomop=10)
It took 5 secs to screen 2430453 sequences, removed 608.

mothur > summary.seqs(fasta=current)

Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 160 160 0 3 1
2.5%-tile: 1 200 200 0 4 60747
25%-tile: 1 203 203 0 5 607462
Median: 1 205 205 0 6 1214923
75%-tile: 1 205 205 0 6 1822384
97.5%-tile: 1 210 210 0 6 2369099
Maximum: 1 230 230 0 10 2429845
Mean: 1 204 204 0 5
# of Seqs: 2429845

It took 17 secs to summarize 2429845 sequences.

mothur > align.seqs(fasta=current, template=/scratch/tnoyes2/scripts/ShortRef.fas)

Reading in the /scratch/tnoyes2/scripts/ShortRef.fas template sequences... DONE.

It took 0 to read 20 sequences.

Aligning sequences from /scratch/tnoyes2/tele02_fastas/tele02.trim.contigs.good.good.fasta ...

It took 39 secs to align 2429845 sequences.

[WARNING]: 58795 of your sequences generated alignments that eliminated too many bases, a list is provided in /scratch/tnoyes2/tele02_fastas/tele02.trim.contigs.good.good.flip.accnos.

[NOTE]: 4920 of your sequences were reversed to produce a better alignment.

It took 39 seconds to align 2429845 sequences.

mothur > screen.seqs(fasta=current, end=182)

It took 6 secs to screen 2429845 sequences, removed 2429845.

mothur > summary.seqs(fasta=current)
tele02.trim.contigs.good.good.good.align as input file for the fasta parameter.
[ERROR]:tele02.trim.contigs.good.good.good.align is blank, aborting.
2

Hi -

It’s most likely that the sequences are getting tossed by the screen.seqs(fasta=current, end=182) statement. Can you post the output of running summary.seqs on the fasta file going into that command (i.e., the aligned sequences)?

Pat

3

Hi Pat, thanks for the reply. sent below

mothur > summary.seqs(fasta=current)
Using tele02_fastas/tele02.trim.contigs.good.good.align as input file for the fasta parameter.

Using 30 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	0	0	0	0	1	1
2.5%-tile:	1	179	166	0	3	60747
25%-tile:	1	179	169	0	5	607462
Median: 	1	179	171	0	6	1214923
75%-tile:	1	179	171	0	6	1822384
97.5%-tile:	1	179	172	0	6	2369099
Maximum:	179	180	180	0	10	2429845
Mean:	5	178	166	0	5
# of Seqs:	2429845
4

I suspect the problem is that in screen.seqs you set end=182, but none of your sequences get to 182. Instead, I’d pick start=1, end=179. Give that a try…

Pat

5

This topic was automatically closed 10 days after the last reply. New replies are no longer allowed.