Warning and error message

1

I was ran Mothur on HPC and received the following warning messages and errors (see below). Have any of you experienced that before? Your assistance is much appreciated. Thanks!

Jean-Rene

mothur > count.seqs(name=current, group=current)

[WARNING]: no file was saved for group parameter.

[WARNING]: no file was saved for name parameter.

You have no current namefile or sharedfile and the name or shared parameter is required, unless inflating or deflating an existing count file.

[ERROR]: did not complete count.seqs.

[WARNING]: 200 of your sequences generated alignments that eliminated too many bases

“[WARNING]: HWI-M03127_118_AHCMU_1_1109_22309_14727 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.

[WARNING]: HWI-M03127_118_AHCMU_1_1108_24018_23579 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.

[WARNING]: HWI-M03127_118_AHCMU_1_2113_16892_10466 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.

[WARNING]: HWI-M03127_118_AHCMU_1_2101_8000_5393 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.

[WARNING]: HWI-M03127_118_AHCMU_1_1110_19686_9112 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.

[WARNING]: HWI-M03127_118_AHCMU_1_2101_20688_27281 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.

[WARNING]: HWI-M03127_118_AHCMU_1_1112_21504_28039 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.

[WARNING]: HWI-M03127_118_AHCMU_1_2103_21763_19913 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.

[WARNING]: HWI-M03127_118_AHCMU_1_1103_13571_18979 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.

[WARNING]: HWI-M03127_118_AHCMU_1_2114_26879_17869 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.”

[WARNING]: Your count file does not have group info, all reads will be assigned to mothurGroup.

0.15

2

Hello Jean-Rene

Without the code you run, it would be very difficult to help you.

Basically, mothur is saying that there are no name or group files assigned as current… So, at some point it dropped them, or they were never assigned.

Could you post the code from the beginning, as you run it?

3

I’m not sure if this is your case, but remember that when you start a new mothur session all of the previous “current” values are forgotten and so you need to manually insert those. Also, if you are using the latest version of mothur, make.contigs will output a count file and you shouldn’t need to run this command.

Pat

4

Hi Leocadio,
Thank you for your response. I ran Mothur on HPC. Here’s the batchfile codes. Thank you for your inputs.
#change the name of the file from stability.files to whatever suits your study

make.contigs(file=/home/u38/S6D14Ana1/S6D14Ana.files, processors=8)

summary.seqs(fasta=current)
screen.seqs(fasta=current, group=current, maxambig=0, maxlength=275)

summary.seqs(fasta=current)
unique.seqs()

count.seqs(name=current, group=current)

summary.seqs(fasta=current, name=current)
#pcr.seqs(fasta=silva.bacteria.fasta, start=11894, end=25319, keepdots=F)

#system(rename silva.bacteria.pcr.fasta silva.v4.fasta)

#summary.seqs()
align.seqs(fasta=current, reference=silva.v4.fasta)

summary.seqs(fasta=current, count=current)
screen.seqs(fasta=current, count=current, start=1968, end=11550, maxhomop=8)

summary.seqs(fasta=current, count=current)
filter.seqs(fasta=current, vertical=T, trump=.)

unique.seqs(fasta=current, count=current)
pre.cluster(fasta=current, count=current, diffs=2)

chimera.uchime(fasta=current, count=current, dereplicate=t)

remove.seqs(fasta=current, accnos=current)
summary.seqs(fasta=current, count=current)
classify.seqs(fasta=current, count=current, reference=trainset9_032012.pds.fasta, taxonomy=trainset9_032012.pds.tax, cutoff=80)

remove.lineage(fasta=current, count=current, taxonomy=current, taxon=Chloroplast-Mitochondria-unknown-Archaea-Eukaryota)

summary.seqs(fasta=current, count=current)
cluster.split(fasta=current, count=current, taxonomy=current, splitmethod=classify, taxlevel=4, cutoff=0.15)

make.shared(list=current, count=current, label=0.03)

classify.otu(list=current, count=current, taxonomy=current, label=0.03, basis=sequence)

5

Hi Pat,
Thank you for your comments. I ran Mothur on HPC, so I had a batchfile. Here are the codes I ran. Your inputs are much appreciated.

#change the name of the file from stability.files to whatever suits your study

make.contigs(file=/home/u38/S6D14Ana1/S6D14Ana.files, processors=8)

summary.seqs(fasta=current)
screen.seqs(fasta=current, group=current, maxambig=0, maxlength=275)

summary.seqs(fasta=current)
unique.seqs()

count.seqs(name=current, group=current)

summary.seqs(fasta=current, name=current)
#pcr.seqs(fasta=silva.bacteria.fasta, start=11894, end=25319, keepdots=F)

#system(rename silva.bacteria.pcr.fasta silva.v4.fasta)

#summary.seqs()
align.seqs(fasta=current, reference=silva.v4.fasta)

summary.seqs(fasta=current, count=current)
screen.seqs(fasta=current, count=current, start=1968, end=11550, maxhomop=8)

summary.seqs(fasta=current, count=current)
filter.seqs(fasta=current, vertical=T, trump=.)

unique.seqs(fasta=current, count=current)
pre.cluster(fasta=current, count=current, diffs=2)

chimera.uchime(fasta=current, count=current, dereplicate=t)

remove.seqs(fasta=current, accnos=current)
summary.seqs(fasta=current, count=current)
classify.seqs(fasta=current, count=current, reference=trainset9_032012.pds.fasta, taxonomy=trainset9_032012.pds.tax, cutoff=80)

remove.lineage(fasta=current, count=current, taxonomy=current, taxon=Chloroplast-Mitochondria-unknown-Archaea-Eukaryota)

summary.seqs(fasta=current, count=current)
cluster.split(fasta=current, count=current, taxonomy=current, splitmethod=classify, taxlevel=4, cutoff=0.15)

make.shared(list=current, count=current, label=0.03)

classify.otu(list=current, count=current, taxonomy=current, label=0.03, basis=sequence)

6

if the problem is in
summary.seqs(fasta=current)
unique.seqs()

count.seqs(name=current, group=current)

you need to do "unique.seqs(fasta=current).

As you have, i doubt that command is working, and no name file is generated. check you logfile for error. Also, not sure about the group file, maybe all goes wrong when not having the name file

7

What version of mothur are you running? If it is the latest version then make.contigs outputs a count file so that you no longer should be running count.seqs

8

unique.seqs() will automatically use the most recently generated fasta file. It’s the same thing as unique.seqs(fasta=current)

Pat

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