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