The align.seqs command is returning a blank file

Hello mothur community. I’m a grad student who is being introduced to bioinformatics, so my familiarity with troubleshooting mothur issues is not the best right now… About a fifth of my samples (reps included) are returning blank align files after I run the align.seqs command, and I’m not too sure why given that the script of code I have works for the majority of my samples. I attached my logfile in the text below, hopefully that will give you guys enough information to work with. The summary.seqs command I used prior to mothur telling me my align file was blank was not included in the logfile. It should also be noted that mothur crashed when I tried to run the command too.

mothur > 
list.seqs(fastq=ETN.20.KN14.swab.3.2_R2.fastq)

Output File Names: 
ETN.20.KN14.swab.3.2_R2.accnos


mothur > 
get.seqs(fastq=ETN.20.KN14.swab.3.2_R1.fastq, accnos=ETN.20.KN14.swab.3.2_R2.accnos)
Selected 243126 sequences from ETN.20.KN14.swab.3.2_R1.fastq.

Output File Names:
ETN.20.KN14.swab.3.2_R1.pick.fastq


mothur > 
list.seqs(fastq=ETN.20.KN14.swab.3.2_R1.pick.fastq)

Output File Names: 
ETN.20.KN14.swab.3.2_R1.pick.accnos


mothur > 
get.seqs(fastq=ETN.20.KN14.swab.3.2_R2.fastq, accnos=ETN.20.KN14.swab.3.2_R1.pick.accnos)
Selected 243126 sequences from ETN.20.KN14.swab.3.2_R2.fastq.

Output File Names:
ETN.20.KN14.swab.3.2_R2.pick.fastq


mothur > 
make.contigs(ffastq = ETN.20.KN14.swab.3.2_R1.pick.fastq, rfastq = ETN.20.KN14.swab.3.2_R2.pick.fastq, trimoverlap =T, insert = 24, deltaq =6, proccessors=1)
[WARNING]: proccessors is not a valid parameter, ignoring.
The valid parameters are: ffastq, rfastq, ffasta, rfasta, fqfile, rqfile, file, oligos, findex, rindex, qfile, pdiffs, bdiffs, tdiffs, checkorient, align, allfiles, trimoverlap, match, mismatch, gapopen, gapextend, insert, deltaq, maxee, processors, format, ksize, maxambig, maxhomop, maxlength, seed, inputdir, and outputdir.

Using 8 processors.
Making contigs...
Done.

It took 32 secs to process 243126 sequences.

Output File Names: 
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.fasta
ETN.20.KN14.swab.3.2_R1.pick.scrap.contigs.fasta
ETN.20.KN14.swab.3.2_R1.pick.contigs_report


mothur > 
summary.seqs(fasta= ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.fasta)

Using 8 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	1	1	0	1	1
2.5%-tile:	1	201	201	0	3	6078
25%-tile:	1	202	202	0	4	60774
Median: 	1	202	202	0	4	121548
75%-tile:	1	202	202	0	4	182321
97.5%-tile:	1	207	207	1	5	237017
Maximum:	1	217	217	53	11	243094
Mean:	1	201	201	0	4
# of Seqs:	243094

It took 1 secs to summarize 243094 sequences.

Output File Names:
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.summary


mothur > 
make.group(fasta=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.fasta, groups=ETN.20.KN14.swab.3.2.W, output=ETN.20.KN14.swab.3.2.groups)

Output File Names: ETN.20.KN14.swab.3.2.groups


mothur > 
screen.seqs(fasta=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.fasta, group=ETN.20.KN14.swab.3.2.groups, maxambig= 0, maxhomop=8)

Using 8 processors.

It took 0 secs to screen 243094 sequences, removed 13074.

/******************************************/
Running command: remove.seqs(accnos=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.bad.accnos.temp, group=ETN.20.KN14.swab.3.2.groups)
Removed 13074 sequences from ETN.20.KN14.swab.3.2.groups.

Output File Names:
ETN.20.KN14.swab.3.2.pick.groups

/******************************************/

Output File Names:
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.fasta
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.bad.accnos
ETN.20.KN14.swab.3.2.good.groups


It took 2 secs to screen 243094 sequences.

mothur > 
summary.seqs(fasta = ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.fasta)

Using 8 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	10	10	0	2	1
2.5%-tile:	1	201	201	0	3	5751
25%-tile:	1	202	202	0	4	57506
Median: 	1	202	202	0	4	115011
75%-tile:	1	202	202	0	4	172516
97.5%-tile:	1	206	206	0	5	224270
Maximum:	1	217	217	0	8	230020
Mean:	1	202	202	0	4
# of Seqs:	230020

It took 1 secs to summarize 230020 sequences.

Output File Names:
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.summary


mothur > 

[ERROR]: You are missing (
[ERROR]: Invalid.

mothur > 
unique.seqs(fasta = ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.fasta)
230020	44523

Output File Names: 
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.fasta
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.count_table


mothur > 
summary.seqs(fasta=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.fasta, count=ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.count_table)

Using 8 processors.

		Start	End	NBases	Ambigs	Polymer	NumSeqs
Minimum:	1	10	10	0	2	1
2.5%-tile:	1	201	201	0	3	5751
25%-tile:	1	202	202	0	4	57506
Median: 	1	202	202	0	4	115011
75%-tile:	1	202	202	0	4	172516
97.5%-tile:	1	206	206	0	5	224270
Maximum:	1	217	217	0	8	230020
Mean:	1	202	202	0	4
# of unique seqs:	44523
total # of seqs:	230020

It took 1 secs to summarize 230020 sequences.

Output File Names:
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.summary


mothur > 
align.seqs(fasta= ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.fasta, reference= silva.nr_v138_1.align)

Using 8 processors.

Reading in the silva.nr_v138_1.align template sequences...	DONE.
It took 156 to read  146601 sequences.

Aligning sequences from ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.fasta ...
It took 109 secs to align 44523 sequences.


It took 117 seconds to align 44523 sequences.

Output File Names: 
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.align
ETN.20.KN14.swab.3.2_R1.pick.trim.contigs.good.unique.align_report

Hi there,

What region of the 16S rRNA gene are you sequencing? What version number of mothur and what operating system are you using?

Pat

We sequenced the V4 region, we’re using mothur v.1.47 ., and we’re using windows 7.

Could you send your fasta file to mothur.bugs@gmail.com so I can take a closer look?

Also, mothur is built for Windows 10. There may be unexpected behavior running it on Windows 7 (release date 2009, support ended 2020). Can you upgrade your version of Windows?

I apologize, sending them to mothur bugs right now

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