An error occurs while running pre.cluster command

Hi

I am running in to a problem with the pre.cluster step.

I am running it as a batch file and the output in mothur log file is as follows.

mothur > summary.seqs(fasta=current)
Using MethodF2.trim.pcr.trim.good.unique.good.filter.unique.fasta as input file for the fasta parameter.

Using 40 processors.

                Start   End     NBases  Ambigs  Polymer NumSeqs
Minimum:        1       589     250     0       3       1
2.5%-tile:      3       802     250     0       3       1182
25%-tile:       3       826     250     0       4       11814
Median:         3       831     250     0       4       23627
75%-tile:       3       835     250     0       5       35440
97.5%-tile:     3       854     250     0       6       46072
Maximum:        3       861     250     0       8       47253
Mean:   2       830     250     0       4
# of Seqs:      47253

It took 1 secs to summarize 47253 sequences.

Output File Names:
MethodF2.trim.pcr.trim.good.unique.good.filter.unique.summary


mothur > pre.cluster(fasta=MethodF2.trim.pcr.trim.good.unique.good.filter.unique.fasta, count=MethodF2.trim.pcr.trim.good.unique.good.filter.count_table, diffs=2)

Using 40 processors.
When using running without group information mothur can only use 1 processor, continuing.
0       47164   89
1000    40802   6451
2000    39346   7907
3000    38493   8760
4000    37873   9380
5000    37356   9897
6000    36952   10301
7000    36918   10335
8000    36918   10335
9000    36918   10335
10000   36918   10335
11000   36918   10335
12000   36918   10335
13000   36918   10335
14000   36918   10335
15000   36918   10335
16000   36918   10335
17000   36918   10335
18000   36918   10335
19000   36918   10335
20000   36918   10335
21000   36918   10335
22000   36918   10335
23000   36918   10335
24000   36918   10335
25000   36918   10335
26000   36918   10335
27000   36918   10335
28000   36918   10335
29000   36918   10335
30000   36918   10335
31000   36918   10335
32000   36918   10335
33000   36918   10335
34000   36918   10335
35000   36918   10335
36000   36918   10335
37000   36918   10335
38000   36918   10335
39000   36918   10335
40000   36918   10335
41000   36918   10335
42000   36918   10335
43000   36918   10335
44000   36918   10335
45000   36918   10335
46000   36918   10335
47000   36918   10335
47253   36918   10335

Total number of sequences before precluster was 47253.
pre.cluster removed 10335 sequences.

/******************************************/
[WARNING]: MethodF2.trim.pcr.trim.good.unique.good.filter.unique.fasta does not contain any sequence from the .accnos file.
Selected 0 sequences from MethodF2.trim.pcr.trim.good.unique.good.filter.unique.fasta.

Output File Names:
MethodF2.trim.pcr.trim.good.unique.good.filter.unique.precluster.fasta

/******************************************/
Done.
It took 282 secs to cluster 47253 sequences.

Using 40 processors.

Output File Names:
MethodF2.trim.pcr.trim.good.unique.good.filter.unique.precluster.fasta
MethodF2.trim.pcr.trim.good.unique.good.filter.unique.precluster.count_table
MethodF2.trim.pcr.trim.good.unique.good.filter.unique.precluster.map

The .fasta out put does not contain any sequences after this step.
Can you please help me with this?

Thanks in advance,
Sam

I seem to be having the same issue with mothur v.1.48.0 so watching this post. I have changed my batch file to reflect the fact that group files are no longer used and am trying to work my way through the issue currently.

it seems this is a redundant issue presented here . I have updated my unique.seqs command to include a count file now and that has solved the issue.

This is the full script that I used up to pre.cluster step.

merge.files(input=BS1.fasta-BS2.fasta, output=MethodF2.fasta)
merge.files(input=BS1.qual-BS2.qual, output=MethodF2.qual)
make.group(fasta=BS1.fasta-BS2.fasta, groups=BS1-BS2, format=group, output=merge.group)
trim.seqs(fasta=MethodF2.fasta, qfile=MethodF2.qual, qwindowaverage=35, qwindowsize=50)
summary.seqs(fasta=current)
pcr.seqs(fasta=MethodF2.trim.fasta, oligos=ForwardOligo.txt)
summary.seqs(fasta=current)
trim.seqs(fasta=MethodF2.trim.pcr.fasta, keepfirst=250)
summary.seqs(fasta=current)
screen.seqs(fasta=MethodF2.trim.pcr.trim.fasta, minlength=250, maxambig=0, maxhomop=8)
summary.seqs(fasta=current)
list.seqs(fasta=MethodF2.trim.pcr.trim.good.fasta)
get.seqs(group=merge.groups, accnos=MethodF2.trim.pcr.trim.good.accnos)
unique.seqs(fasta=MethodF2.trim.pcr.trim.good.fasta, count=current)
align.seqs(fasta=MethodF2.trim.pcr.trim.good.unique.fasta, reference=silva.nr_v138.r678.align, flip=F)
screen.seqs(fasta=MethodF2.trim.pcr.trim.good.unique.align, count=MethodF2.trim.pcr.trim.good.count_table, minlength=250, start= 809, end=9000)
filter.seqs(fasta=MethodF2.trim.pcr.trim.good.unique.good.align, vertical=T)
summary.seqs(fasta=current)
unique.seqs(fasta=MethodF2.trim.pcr.trim.good.unique.good.filter.fasta, count=MethodF2.trim.pcr.trim.good.good.count_table)
summary.seqs(fasta=current)
pre.cluster(fasta=MethodF2.trim.pcr.trim.good.unique.good.filter.unique.fasta, count=MethodF2.trim.pcr.trim.good.unique.good.filter.count_table, diffs=2)

Still the output .fasta file contains 0 sequences.

I’m having a hard time following your pipeline or understanding what you are trying to do. Can you annotate it? I notice you make a group file in the third command but you don’t use it in any of the screen.seqs commands. It’s also not clear where the count file was generated.

Pat

Hi Pat,

Apologies… I have not put one code in the middle in the script I posted here. After the unique.seq command I use count.seqs(name=.names, group=.groups) file to generate a count table. However, in the new versions of mothur, .names file and .groups file is not given as an output. This script was taken from a published paper that used mothur v.1.43. I tried the older version of mothur with the script and it worked.
But for future purpose, Do you have any advice?

Thanks,
Sam

Have you looked at the SOP? It has been updated for the newer version of mothur that doesn’t use names and groups files.

Pat