Hello I am trying to use mothur to analyze samples from region V3-V4. I’m new to microbiome data analysis, but I was able to complete all the steps of the MiSeq SOP on the V4 region provided as a test dataset and everything is working correctly.
Based on the MiSeq SOP, I created a pipeline to analyze our own sequencing data, only changing the region coordinates as described here Pcr.seqs: How do you know where your sequences start and end against the reference database? · Issue #235 · mothur/mothur · GitHub. Unfortunately, I’ve encountered an error that I can’t handle. This happens in the unique.seqs step - mothur can’t open the count file. I found out that this file does not actually exist, since it was not created at the screen.seqs step. No other errors are reported and I have no idea what is wrong and what I should do to resolve this issue and continue with the analysis. I hope you give me a hand. I’m using mothur v.1.44.3, below is the mothur log I get.
mothur > 2022-01-11.batch
/*****************************************************************************/
mothur > make.file(inputdir=., type=fastq, prefix=2022-01-11)
Setting input directory to: /home/my/dir
[WARNNG]: mothur found unpaired files in your input directory. Outputting list of filenames to /home/my/dir/2022-01-11.single.files for your review.
Output File Names:
/home/my/dir/2022-01-11.single.files
/home/my/dir/2022-01-11.files
mothur > make.contigs(file=2022-01-11.files, processors=8)
Using 8 processors.
Making contigs…
Done.
Group count:
2F002 6078
3F002 5186
4F002 5331
6F003 6658
Mock 4493
Total of all groups is 27746
It took 3 secs to process 27746 sequences.
Output File Names:
/home/my/dir/2022-01-11.trim.contigs.fasta
/home/my/dir/2022-01-11.scrap.contigs.fasta
/home/my/dir/2022-01-11.contigs.report
/home/my/dir/2022-01-11.contigs.groups
mothur > summary.seqs(fasta=2022-01-11.trim.contigs.fasta)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 242 242 0 3 1
2.5%-tile: 1 439 439 0 4 694
25%-tile: 1 440 440 0 5 6937
Median: 1 442 442 0 5 13874
75%-tile: 1 460 460 0 5 20810
97.5%-tile: 1 466 466 0 6 27053
Maximum: 1 467 467 5 16 27746
Mean: 1 448 448 0 5
of Seqs: 27746
It took 0 secs to summarize 27746 sequences.
Output File Names:
/home/my/dir/2022-01-11.trim.contigs.summary
mothur > screen.seqs(fasta=2022-01-11.trim.contigs.fasta, group=2022-01-11.contigs.groups, maxambig=0, maxlength=467, maxhomop=6)
Using 8 processors.
It took 0 secs to screen 27746 sequences, removed 938.
/******************************************/
Running command: remove.seqs(accnos=/home/my/dir/2022-01-11.trim.contigs.bad.accnos.temp, group=/home/my/dir/2022-01-11.contigs.groups)
Removed 938 sequences from your group file.
Output File Names:
/home/my/dir/2022-01-11.contigs.pick.groups
/******************************************/
Output File Names:
/home/my/dir/2022-01-11.trim.contigs.good.fasta
/home/my/dir/2022-01-11.trim.contigs.bad.accnos
/home/my/dir/2022-01-11.contigs.good.groups
mothur> count.seqs(name=2022-01-11.trim.contigs.good.names, group=2022-01-11.contigs.good.groups)
It took 0 secs to screen 27746 sequences.
26808 23594
Output File Names:
/home/my/dir/2022-01-11.trim.contigs.good.names
/home/my/dir/2022-01-11.trim.contigs.good.unique.fasta
mothur > count.seqs(name=2022-01-11.trim.contigs.good.names, group=2022-01-11.contigs.good.groups)
It took 0 secs to create a table for 26808 sequences.
Total number of sequences: 26808
Output File Names:
/home/my/dir/2022-01-11.trim.contigs.good.count_table
mothur > pcr.seqs(fasta=silva.bacteria.fasta, start=6428, end=23440, keepdots=F, processors=8)
Using 8 processors.
[NOTE]: no sequences were bad, removing /home/my/dir/silva.bacteria.bad.accnos
It took 3 secs to screen 14956 sequences.
Output File Names:
/home/my/dir/silva.bacteria.pcr.fasta
mothur > rename.file(input=silva.bacteria.pcr.fasta, new=silva.v3v4.fasta)
Current files saved by mothur:
accnos=/home/my/dir/2022-01-11.trim.contigs.bad.accnos
fasta=/home/my/dir/silva.bacteria.pcr.fasta
group=/home/my/dir/2022-01-11.contigs.good.groups
name=/home/my/dir/2022-01-11.trim.contigs.good.names
contigsreport=/home/my/dir/2022-01-11.contigs.report
count=/home/my/dir/2022-01-11.trim.contigs.good.count_table
processors=8
summary=/home/my/dir/2022-01-11.trim.contigs.summary
file=/home/my/dir/2022-01-11.files
mothur > summary.seqs(fasta=silva.v3v4.fasta)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 16155 381 0 3 1
2.5%-tile: 2 17012 401 0 4 374
25%-tile: 2 17012 404 0 4 3740
Median: 2 17012 423 0 5 7479
75%-tile: 2 17012 426 0 5 11218
97.5%-tile: 2 17012 427 1 6 14583
Maximum: 8 17012 460 5 9 14956
Mean: 2 17011 416 0 4
of Seqs: 14956
It took 1 secs to summarize 14956 sequences.
Output File Names:
/home/my/dir/silva.v3v4.summary
mothur > align.seqs(fasta=2022-01-11.trim.contigs.good.unique.fasta, reference=silva.v3v4.fasta)
Using 8 processors.
Reading in the /home/my/dir/silva.v3v4.fasta template sequences… DONE.
It took 3 to read 14956 sequences.
Aligning sequences from /home/my/dir/2022-01-11.trim.contigs.good.unique.fasta …
It took 7 secs to align 23594 sequences.
It took 7 seconds to align 23594 sequences.
Output File Names:
/home/my/dir/2022-01-11.trim.contigs.good.unique.align
/home/my/dir/2022-01-11.trim.contigs.good.unique.align.report
mothur > summary.seqs(fasta=2022-01-11.trim.contigs.good.unique.align, count=2022-01-11.trim.contigs.good.count_table)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 17012 219 0 3 1
2.5%-tile: 2 17012 401 0 4 671
25%-tile: 2 17012 401 0 4 6703
Median: 2 17012 403 0 5 13405
75%-tile: 2 17012 421 0 5 20107
97.5%-tile: 2 17012 427 0 6 26138
Maximum: 8557 17012 427 0 6 26808
Mean: 8 17012 409 0 4
of unique seqs: 23594
total # of seqs: 26808
It took 1 secs to summarize 26808 sequences.
Output File Names:
/home/my/dir/2022-01-11.trim.contigs.good.unique.summary
mothur > screen.seqs(fasta=2022-01-11.trim.contigs.good.unique.align, count=2022-01-11.trim.contigs.good.count_table, summary=2022-01-11.trim.contigs.good.unique.summary, start=8557, end=17012, maxhomop=8)
Using 8 processors.
It took 0 secs to screen 23594 sequences, removed 0.
[NOTE]: no sequences were bad, removing /home/my/dir/2022-01-11.trim.contigs.good.unique.bad.accnos
Output File Names:
/home/my/dir/2022-01-11.trim.contigs.good.unique.good.summary
/home/my/dir/2022-01-11.trim.contigs.good.unique.good.align
It took 0 secs to screen 23594 sequences.
mothur > summary.seqs(fasta=current, count=current)
Using /home/my/dir/2022-01-11.trim.contigs.good.count_table as input file for the count parameter.
Using /home/my/dir/2022-01-11.trim.contigs.good.unique.good.align as input file for the fasta parameter.
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 17012 219 0 3 1
2.5%-tile: 2 17012 401 0 4 671
25%-tile: 2 17012 401 0 4 6703
Median: 2 17012 403 0 5 13405
75%-tile: 2 17012 421 0 5 20107
97.5%-tile: 2 17012 427 0 6 26138
Maximum: 8557 17012 427 0 6 26808
Mean: 8 17012 409 0 4
of unique seqs: 23594
total # of seqs: 26808
It took 1 secs to summarize 26808 sequences.
Output File Names:
/home/my/dir/2022-01-11.trim.contigs.good.unique.good.summary
mothur > filter.seqs(fasta=2022-01-11.trim.contigs.good.unique.good.align, vertical=T, trump=.)
Using 8 processors.
Creating Filter…
It took 1 secs to create filter for 23594 sequences.
Running Filter…
It took 1 secs to filter 23594 sequences.
Length of filtered alignment: 280
Number of columns removed: 16732
Length of the original alignment: 17012
Number of sequences used to construct filter: 23594
Output File Names:
/home/my/dir/2022-01-11.filter
/home/my/dir/2022-01-11.trim.contigs.good.unique.good.filter.fasta
mothur > unique.seqs(fasta=2022-01-11.trim.contigs.good.unique.good.filter.fasta, count=2022-01-11.trim.contigs.good.good.count_table)
Unable to open /home/my/dir/2022-01-11.trim.contigs.good.good.count_table. Trying input directory /home/my/dir/2022-01-11.trim.contigs.good.good.count_table.
Unable to open /home/my/dir/2022-01-11.trim.contigs.good.good.count_table. Trying default /usr/bin//2022-01-11.trim.contigs.good.good.count_table.
Unable to open /usr/bin//2022-01-11.trim.contigs.good.good.count_table. Trying mothur’s executable location /usr/bin//2022-01-11.trim.contigs.good.good.count_table.
Unable to open /usr/bin//2022-01-11.trim.contigs.good.good.count_table. Trying mothur’s tools location 2022-01-11.trim.contigs.good.good.count_table.
Unable to open 2022-01-11.trim.contigs.good.good.count_table.
Unable to open /home/my/dir/2022-01-11.trim.contigs.good.good.count_table
[WARNING]: This command can take a namefile and you did not provide one. The current namefile is /home/my/dir/2022-01-11.trim.contigs.good.names which seems to match /home/my/dir/2022-01-11.trim.contigs.good.unique.good.filter.fasta.
[ERROR]: did not complete unique.seqs.
mothur > pre.cluster(fasta=2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta, count=2022-01-11.trim.contigs.good.unique.good.filter.count_table, diffs=2)
Unable to open /home/my/dir/2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta. Trying input directory /home/my/dir/2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta.
Unable to open /home/my/dir/2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta. Trying default /usr/bin//2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta.
Unable to open /usr/bin//2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta. Trying mothur’s executable location /usr/bin//2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta.
Unable to open /usr/bin//2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta. Trying mothur’s tools location 2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta.
Unable to open 2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta.
Unable to open /home/my/dir/2022-01-11.trim.contigs.good.unique.good.filter.unique.fasta
Unable to open /home/my/dir/2022-01-11.trim.contigs.good.unique.good.filter.count_table. Trying input directory /home/my/dir/2022-01-11.trim.contigs.good.unique.good.filter.count_table.
Unable to open/home/my/dir/2022-01-11.trim.contigs.good.unique.good.filter.count_table. Trying default /usr/bin//2022-01-11.trim.contigs.good.unique.good.filter.count_table.
Unable to open /usr/bin//2022-01-11.trim.contigs.good.unique.good.filter.count_table. Trying mothur’s executable location /usr/bin//2022-01-11.trim.contigs.good.unique.good.filter.count_table.
Unable to open /usr/bin//2022-01-11.trim.contigs.good.unique.good.filter.count_table. Trying mothur’s tools location 2022-01-11.trim.contigs.good.unique.good.filter.count_table.
Unable to open 2022-01-11.trim.contigs.good.unique.good.filter.count_table.
Unable to open /home/my/dir/seq11_01_22/2022-01-11.trim.contigs.good.unique.good.filter.count_table