Hello, @SRC_151
I had a similar issue when trying to remove chimeras
“[ERROR]: Could not open stability.trim.contigs.good.unique.good.temp
Error in reading your fastafile, at position -1. Blank name.) but was using the UCHIME command (chimera.uchime(fasta=stability.trim.contigs.good.unique.good.align, reference=silva.v4.fasta)”
I believe I “fixed” this by changing “reference=” to “count=current”.
This may be 100% wrong, and I may be realizing unintended results, but the “count” option looks very similar in mothur’s vsearch and uchime.
I would wait for a more professional opinion (@pschloss)before charging ahead with your analysis, but this change cleared the error.
See below for the logfile (the last bit is relevant). I apologise if this is an improper drop of a log file, this is the first time I’ve ever contributed to a forum on any site. Please advise if anything is below board! 
Linux version
Using Boost,HDF5,GSL
mothur v.1.48.3
Last updated: 5/21/25
by
Patrick D. Schloss
Department of Microbiology & Immunology
University of Michigan
http://www.mothur.org
When using, please cite:
Schloss, P.D., et al., Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol, 2009. 75(23):7537-41.
Distributed under the GNU General Public License
Type 'help()' for information on the commands that are available
For questions and analysis support, please visit our forum at https://forum.mothur.org
Type 'quit()' to exit program
[NOTE]: Setting random seed to 19760620.
Interactive Mode
mothur >
mothur > make.file(inputdir=., type=fastq, prefix=stability)
Setting input directories to:
<usr wd>
Output File Names:
<usr wd>stability.files
mothur >
mothur > make.contigs(file=current, maxambig=0, maxlength=500, maxhomop=8, oligos=primers.oligo, pdiffs=0, insert=30)
Using <usr wd>stability.files as input file for the file parameter.
Using 16 processors.
>>>>> Processing file pair ERR2179951_1.fastq - ERR2179951_2.fastq (files 1 of 7) <<<<<
Making contigs...
Done.
It took 17 secs to assemble 192608 reads.
>>>>> Processing file pair ERR2179952_1.fastq - ERR2179952_2.fastq (files 2 of 7) <<<<<
Making contigs...
Done.
It took 11 secs to assemble 168659 reads.
>>>>> Processing file pair ERR2179953_1.fastq - ERR2179953_2.fastq (files 3 of 7) <<<<<
Making contigs...
Done.
It took 12 secs to assemble 170196 reads.
>>>>> Processing file pair ERR2179954_1.fastq - ERR2179954_2.fastq (files 4 of 7) <<<<<
Making contigs...
Done.
It took 8 secs to assemble 129539 reads.
>>>>> Processing file pair ERR2179955_1.fastq - ERR2179955_2.fastq (files 5 of 7) <<<<<
Making contigs...
Done.
It took 11 secs to assemble 148287 reads.
>>>>> Processing file pair ERR2179956_1.fastq - ERR2179956_2.fastq (files 6 of 7) <<<<<
Making contigs...
Done.
It took 7 secs to assemble 103859 reads.
>>>>> Processing file pair ERR2179957_1.fastq - ERR2179957_2.fastq (files 7 of 7) <<<<<
Making contigs...
Done.
It took 19 secs to assemble 279553 reads.
Group count:
ERR2179951 134480
ERR2179952 121728
ERR2179953 121072
ERR2179954 92847
ERR2179955 105730
ERR2179956 75469
ERR2179957 204189
Total of all groups is 855515
It took 90 secs to process 1192701 sequences.
Output File Names:
<usr wd>stability.trim.contigs.fasta
<usr wd>stability.scrap.contigs.fasta
<usr wd>stability.contigs_report
<usr wd>stability.contigs.count_table
mothur >
mothur > summary.seqs(fasta=current)
Using <usr wd>stability.trim.contigs.fasta as input file for the fasta parameter.
Using 16 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 237 237 0 3 1
2.5%-tile: 1 400 400 0 4 21388
25%-tile: 1 400 400 0 4 213879
Median: 1 420 420 0 4 427758
75%-tile: 1 425 425 0 5 641637
97.5%-tile: 1 425 425 0 6 834128
Maximum: 1 463 463 0 8 855515
Mean: 1 415 415 0 4
# of Seqs: 855515
It took 2 secs to summarize 855515 sequences.
Output File Names:
<usr wd>stability.trim.contigs.summary
mothur >
mothur > screen.seqs(fasta=stability.trim.contigs.fasta, count=stability.contigs.count_table, maxambig=0, maxlength=500, minlength=300, maxhomop=8, contigsreport=stability.contigs_report, minoverlap=30, mismatches=0)
Using 16 processors.
It took 5 secs to screen 855515 sequences, removed 506397.
/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.bad.accnos.temp, count=stability.contigs.count_table)
Removed 506397 sequences from stability.contigs.count_table.
Output File Names:
stability.contigs.pick.count_table
/******************************************/
Output File Names:
stability.good.[extension]
stability.trim.contigs.good.fasta
stability.trim.contigs.bad.accnos
stability.contigs.good.count_table
It took 25 secs to screen 855515 sequences.
mothur >
mothur > summary.seqs(fasta=current)
Using stability.trim.contigs.good.fasta as input file for the fasta parameter.
Using 16 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 300 300 0 3 1
2.5%-tile: 1 400 400 0 4 8728
25%-tile: 1 400 400 0 4 87280
Median: 1 420 420 0 4 174560
75%-tile: 1 425 425 0 5 261839
97.5%-tile: 1 425 425 0 6 340391
Maximum: 1 434 434 0 8 349118
Mean: 1 414 414 0 4
# of Seqs: 349118
It took 1 secs to summarize 349118 sequences.
Output File Names:
stability.trim.contigs.good.summary
mothur >
mothur > unique.seqs(fasta=stability.trim.contigs.good.fasta, count=stability.contigs.good.count_table)
349118 119999
Output File Names:
stability.trim.contigs.good.unique.fasta
stability.trim.contigs.good.count_table
mothur >
mothur > pcr.seqs(fasta=silva.nr_v138_2.align, start=6388, end=25293, keepdots=F)
Using 16 processors.
[NOTE]: no sequences were bad, removing silva.nr_v138_2.bad.accnos
It took 19 secs to screen 164296 sequences.
Output File Names:
silva.nr_v138_2.pcr.align
mothur >
mothur > rename.file(input=silva.nr_v138_2.pcr.align, new=silva.v4.fasta)
Current files saved by mothur:
accnos=stability.trim.contigs.bad.accnos
fasta=silva.nr_v138_2.pcr.align
oligos=primers.oligo
contigsreport=<usr wd>stability.contigs_report
count=stability.trim.contigs.good.count_table
processors=16
summary=stability.trim.contigs.good.summary
file=<usr wd>stability.files
mothur >
mothur > align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.v4.fasta)
Using 16 processors.
Reading in the silva.v4.fasta template sequences... DONE.
It took 46 to read 164296 sequences.
Aligning sequences from stability.trim.contigs.good.unique.fasta ...
It took 197 secs to align 119999 sequences.
[WARNING]: 2 of your sequences generated alignments that eliminated too many bases, a list is provided in stability.trim.contigs.good.unique.flip.accnos.
[NOTE]: 1 of your sequences were reversed to produce a better alignment.
It took 197 seconds to align 119999 sequences.
Output File Names:
stability.trim.contigs.good.unique.align
stability.trim.contigs.good.unique.align_report
stability.trim.contigs.good.unique.flip.accnos
mothur >
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: 1 15 2 0 1 1
2.5%-tile: 41 16201 400 0 4 8728
25%-tile: 41 16201 400 0 4 87280
Median: 41 16201 420 0 4 174560
75%-tile: 41 16201 425 0 5 261839
97.5%-tile: 41 16201 425 0 6 340391
Maximum: 18905 18906 434 0 8 349118
Mean: 49 16200 414 0 4
# of unique seqs: 119999
total # of seqs: 349118
It took 8 secs to summarize 349118 sequences.
Output File Names:
stability.trim.contigs.good.unique.summary
mothur >
mothur > screen.seqs(fasta=current, count=current, start=41, end=16201)
Using stability.trim.contigs.good.count_table as input file for the count parameter.
Using stability.trim.contigs.good.unique.align as input file for the fasta parameter.
Using 16 processors.
It took 7 secs to screen 119999 sequences, removed 1727.
/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.good.unique.bad.accnos.temp, count=stability.trim.contigs.good.count_table)
Removed 2320 sequences from stability.trim.contigs.good.count_table.
Output File Names:
stability.trim.contigs.good.pick.count_table
/******************************************/
Output File Names:
stability.trim.contigs.good.unique.good.align
stability.trim.contigs.good.unique.bad.accnos
stability.trim.contigs.good.good.count_table
It took 10 secs to screen 119999 sequences.
mothur >
mothur > filter.seqs(fasta=current, vertical=T, trump=.)
Using stability.trim.contigs.good.unique.good.align as input file for the fasta parameter.
Using 16 processors.
Creating Filter...
It took 8 secs to create filter for 118272 sequences.
Running Filter...
It took 3 secs to filter 118272 sequences.
Length of filtered alignment: 811
Number of columns removed: 18095
Length of the original alignment: 18906
Number of sequences used to construct filter: 118272
Output File Names:
stability.filter
stability.trim.contigs.good.unique.good.filter.fasta
mothur >
mothur > unique.seqs(fasta=current, count=current)
Using stability.trim.contigs.good.good.count_table as input file for the count parameter.
Using stability.trim.contigs.good.unique.good.filter.fasta as input file for the fasta parameter.
118272 118255
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.fasta
stability.trim.contigs.good.unique.good.filter.count_table
mothur >
mothur > pre.cluster(fasta=current, count=current, diffs=4)
Using stability.trim.contigs.good.unique.good.filter.count_table as input file for the count parameter.
Using stability.trim.contigs.good.unique.good.filter.unique.fasta as input file for the fasta parameter.
Using 16 processors.
/******************************************/
Splitting by sample:
Using 16 processors.
Reducing processors to 7.
Selecting sequences for groups ERR2179956
Selecting sequences for groups ERR2179954
Selecting sequences for groups ERR2179953
Selecting sequences for groups ERR2179955
Selecting sequences for groups ERR2179951
Selecting sequences for groups ERR2179952
Selecting sequences for groups ERR2179957
Selected 18061 sequences from ERR2179951.
Selected 15470 sequences from ERR2179954.
Selected 18349 sequences from ERR2179953.
Selected 13731 sequences from ERR2179956.
Selected 21904 sequences from ERR2179952.
Selected 17752 sequences from ERR2179955.
Selected 35572 sequences from ERR2179957.
It took 5 seconds to split the dataset by sample.
/******************************************/
Reducing processors to 7.
Processing group ERR2179952:
Processing group ERR2179953:
Processing group ERR2179954:
Processing group ERR2179955:
Processing group ERR2179956:
Processing group ERR2179957:
Processing group ERR2179951:
ERR2179951 18061 1917 16144
Total number of sequences before pre.cluster was 18061.
pre.cluster removed 16144 sequences.
It took 2 secs to cluster 18061 sequences.
ERR2179956 13731 3757 9974
Total number of sequences before pre.cluster was 13731.
pre.cluster removed 9974 sequences.
It took 3 secs to cluster 13731 sequences.
ERR2179953 18349 3615 14734
Total number of sequences before pre.cluster was 18349.
pre.cluster removed 14734 sequences.
It took 3 secs to cluster 18349 sequences.
ERR2179954 15470 3795 11675
Total number of sequences before pre.cluster was 15470.
pre.cluster removed 11675 sequences.
It took 3 secs to cluster 15470 sequences.
ERR2179955 17752 4641 13111
Total number of sequences before pre.cluster was 17752.
pre.cluster removed 13111 sequences.
It took 3 secs to cluster 17752 sequences.
ERR2179952 21904 5586 16318
Total number of sequences before pre.cluster was 21904.
pre.cluster removed 16318 sequences.
It took 4 secs to cluster 21904 sequences.
ERR2179957 35572 9520 26052
Total number of sequences before pre.cluster was 35572.
pre.cluster removed 26052 sequences.
It took 7 secs to cluster 35572 sequences.
Deconvoluting count table results...
It took 0 secs to merge 32831 sequences group data.
/******************************************/
Running get.seqs:
Selected 30197 sequences from stability.trim.contigs.good.unique.good.filter.unique.fasta.
/******************************************/
It took 14 secs to run pre.cluster.
Using 16 processors.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.ERR2179951.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.ERR2179952.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.ERR2179953.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.ERR2179954.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.ERR2179955.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.ERR2179956.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.ERR2179957.map
mothur >
mothur > get.current()
Current RAM usage: 5.97609 Gigabytes. Total Ram: 60.6232 Gigabytes.
Current files saved by mothur:
accnos=stability.trim.contigs.good.unique.bad.accnos
fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta
oligos=primers.oligo
contigsreport=<usr wd>stability.contigs_report
count=stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table
processors=16
summary=stability.trim.contigs.good.unique.summary
file=<usr wd>stability.files
Current input directories saved by mothur:
<usr wd>
Current working directory: <usr wd>
Output File Names:
current_files.summary
mothur >
mothur > chimera.uchime(fasta=current, reference=silva.v4.fasta, name=chimeras.fasta)
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta as input file for the fasta parameter.
Unable to open chimeras.fasta. Trying input directory <usr wd>chimeras.fasta.
Unable to open <usr wd>chimeras.fasta. Trying mothur's executable directory chimeras.fasta.
Unable to open chimeras.fasta.
Unable to open chimeras.fasta
Using 16 processors.
Unable to open uchime. Trying input directory <usr wd>uchime.
Unable to open <usr wd>uchime. Trying mothur's executable directory uchime.
Unable to open uchime.
uchime file does not exist. Checking path...
Found uchime in your path, using /home/<usr>/mothur/Mothur.linux_8_noReadline//uchime
[ERROR]: did not complete chimera.uchime.
mothur >
mothur > chimera.uchime(fasta=current, reference=silva.v4.fasta)
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta as input file for the fasta parameter.
Using 16 processors.
Unable to open uchime. Trying input directory <usr wd>uchime.
Unable to open <usr wd>uchime. Trying mothur's executable directory uchime.
Unable to open uchime.
uchime file does not exist. Checking path...
Found uchime in your path, using /home/<usr>/mothur/Mothur.linux_8_noReadline//uchime
uchime by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.
Checking sequences from stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta ...
[ERROR]: Could not open stability.trim.contigs.good.unique.good.filter.unique.precluster.temp
Error in reading your fastafile, at position -1. Blank name.
mothur >
mothur > chimera.uchime(fasta=current, reference=silv
[ERROR]: You are missing )
[ERROR]: Invalid.
mothur >
mothur > chimera.uchime(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, reference=silva.v4.fasta)
Using 16 processors.
Unable to open uchime. Trying input directory <usr wd>uchime.
Unable to open <usr wd>uchime. Trying mothur's executable directory uchime.
Unable to open uchime.
uchime file does not exist. Checking path...
Found uchime in your path, using /home/<usr>/mothur/Mothur.linux_8_noReadline//uchime
uchime by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.
Checking sequences from stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta ...
[ERROR]: Could not open stability.trim.contigs.good.unique.good.filter.unique.precluster.temp
Error in reading your fastafile, at position -1. Blank name.
mothur >
mothur > chimera.uchime(fasta=stability.trim.contigs.good.unique.good.align, reference=silva.v4.fasta)
Using 16 processors.
Unable to open uchime. Trying input directory <usr wd>uchime.
Unable to open <usr wd>uchime. Trying mothur's executable directory uchime.
Unable to open uchime.
uchime file does not exist. Checking path...
Found uchime in your path, using /home/<usr>/mothur/Mothur.linux_8_noReadline//uchime
uchime by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.
Checking sequences from stability.trim.contigs.good.unique.good.align ...
[ERROR]: Could not open stability.trim.contigs.good.unique.good.temp
Error in reading your fastafile, at position -1. Blank name.
mothur >
mothur > chimera.uchime(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=current)
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table as input file for the count parameter.
Using 16 processors.
Unable to open uchime. Trying input directory <usr wd>uchime.
Unable to open <usr wd>uchime. Trying mothur's executable directory uchime.
Unable to open uchime.
uchime file does not exist. Checking path...
Found uchime in your path, using /home/<usr>/mothur/Mothur.linux_8_noReadline//uchime
uchime by Robert C. Edgar
http://drive5.com/uchime
This code is donated to the public domain.
Checking sequences from stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta ...
/******************************************/
Splitting by sample:
Using 16 processors.
Reducing processors to 7.
Selecting sequences for groups ERR2179951
Selecting sequences for groups ERR2179953
Selecting sequences for groups ERR2179954
Selecting sequences for groups ERR2179956
Selecting sequences for groups ERR2179955
Selecting sequences for groups ERR2179952
Selecting sequences for groups ERR2179957
Selected 3795 sequences from ERR2179954.
Selected 3757 sequences from ERR2179956.
Selected 1917 sequences from ERR2179951.
Selected 3615 sequences from ERR2179953.
Selected 4641 sequences from ERR2179955.
Selected 9520 sequences from ERR2179957.
Selected 5586 sequences from ERR2179952.
It took 2 seconds to split the dataset by sample.
/******************************************/
Reducing processors to 7.
It took 24 secs to check 1917 sequences from group ERR2179951.
It took 53 secs to check 3795 sequences from group ERR2179954.
It took 57 secs to check 3615 sequences from group ERR2179953.
It took 58 secs to check 3757 sequences from group ERR2179956.
It took 68 secs to check 4641 sequences from group ERR2179955.
It took 90 secs to check 5586 sequences from group ERR2179952.
It took 217 secs to check 9520 sequences from group ERR2179957.
It took 217 secs to check 32831 sequences.
It took 220 secs to check 32831 sequences. 16159 chimeras were found.
The number of sequences checked may be larger than the number of unique sequences because some sequences are found in several samples.
Removing chimeras from your input files:
/******************************************/
Running command: remove.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, accnos=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.accnos, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table)
Removed 16159 sequences from stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta.
Removed 35355 sequences from stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.count_table
/******************************************/
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.chimeras
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.accnos
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.fasta