# Cluster.split issue (again, sorry)

**URL:** <https://forum.mothur.org/t/cluster-split-issue-again-sorry/21196>\
**Category:** mothur bugs\
**Created:** [November 29, 2021, 1:32pm UTC](https://forum.mothur.org/t/cluster-split-issue-again-sorry/21196 "2021-11-29T13:32:32Z")\
**Posts on this page:** 5\
**Page:** 1

<div class="post-metadata">

**Author:** ![Alexandre\_Thibodeau](https://avatars.discourse-cdn.com/v4/letter/a/e480ec/32.png) [@Alexandre\_Thibodeau](https://forum.mothur.org/u/Alexandre_Thibodeau)\
**Post date:** [November 29, 2021, 1:32pm UTC](https://forum.mothur.org/t/cluster-split-issue-again-sorry/21196/1 "2021-11-29T13:32:32Z")

</div>

Hello!

This is not my first time asking about this, but this time around I am out of options. I need to run cluster.split (because when I run normal clustering I am loosing sequences in my positive control, namely my Salmonella unless I cluster at 0.005) So I want to use cluster.split to make sure that my positive control says as it should be and cluster everything under at 0.03.

But, when I run it, I get many error messages: "Your group file contains 390863 sequences and list file contains 390790 sequences. Please correct.  
"

Which does not make sense to me since I am running my batch file using “current” for all files, therefore all files are correctly used. I am using 32 thread, is this a problem with using multiple or too much paralleling? I need to run around 700 samples together. Thanks for your input. Here is the spec I am using to run the samples (it takes me 23 hours to run, version v.1.44.3 which is the latest available on the computer servers I use) and my batch file.

Kind regards,

mem=128000M  
cpus-per-task=32

set.current(processors=32)

set.logfile(name=megacampy\_logFile\_clustersplit)

make.contigs(file=megacampy.files, oligos=primers.oligo.txt, checkorient=t, pdiffs=0, maxee=2,deltaq=6)  
summary.seqs(fasta=current)

screen.seqs(fasta=current, group=current, summary=current, maxambig=0, maxhomop=70)  
count.groups(group=current)  
summary.seqs(fasta=current)

unique.seqs(fasta=current)  
count.seqs(name=current, group=current)  
summary.seqs(fasta=current, count=current)

align.seqs(fasta=current, reference=silva.nr\_v132.pcr.align, flip=t)  
summary.seqs(fasta=current, count=current)  
count.groups(group=current)

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

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

pre.cluster(fasta=current, count=current, diffs=4)  
count.groups(count=current)  
summary.seqs(fasta=current, count=current)

chimera.vsearch(fasta=current, count=current, dereplicate=t)  
remove.seqs(fasta=current, accnos=current)  
count.groups(count=current)  
summary.seqs(fasta=current, count=current)

classify.seqs(fasta=current, count=current, iters=1000, reference=silva.nr\_v138.align, taxonomy=silva.nr\_v138.tax, cutoff=80)

remove.lineage(fasta=current, count=current, taxonomy=current, taxon=Chloroplast-Mitochondria-unknown-Eukaryota)  
summary.tax(taxonomy=current, count=current)  
summary.seqs(fasta=current, count=current)  
count.groups(count=current)

dist.seqs(fasta=current,cutoff=0.04)

cluster.split(column=current, count=current, taxonomy=current, splitmethod=classify, taxlevel=6, delta=0, iters=500, cutoff=0.03)

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

classify.otu(list=current, count=current, taxonomy=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Positif\_PCR\_sophie)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Zymo\_Ctrl\_PCR\_carvacrol)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=zymo\_extraction\_carvacrol)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Zymo\_community\_sophie)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Positif\_PCR2\_sophie)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Commart\_probio)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=campy\_protease\_Zymo\_extraction)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

quit()

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<div class="post-metadata">

**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\
**Post date:** [November 30, 2021, 6:17pm UTC](https://forum.mothur.org/t/cluster-split-issue-again-sorry/21196/2 "2021-11-30T18:17:11Z")

</div>

Could you please email your log file and a link to this post to [mothur.bugs@gmail.com](mailto:mothur.bugs@gmail.com)?

Thanks,  
Pat

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<div class="post-metadata">

**Author:** ![Alexandre\_Thibodeau](https://avatars.discourse-cdn.com/v4/letter/a/e480ec/32.png) [@Alexandre\_Thibodeau](https://forum.mothur.org/u/Alexandre_Thibodeau)\
**Post date:** [November 30, 2021, 9:41pm UTC](https://forum.mothur.org/t/cluster-split-issue-again-sorry/21196/3 "2021-11-30T21:41:06Z")

</div>

thanks!

I was able to get 1.46.1, rerunning it as for now.

But, while checking around the forum, I wondered if this could be a problem with some sequence names such as : “M06648\_144\_000000000-JKPRF\_1\_2105\_10757\_18173” that contain the character “-”?  
I spotted this sequence in my server output (I am at the classification step, should have final results by the end of tomorow).

Cheers!

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<div class="post-metadata">

**Author:** ![Alexandre\_Thibodeau](https://avatars.discourse-cdn.com/v4/letter/a/e480ec/32.png) [@Alexandre\_Thibodeau](https://forum.mothur.org/u/Alexandre_Thibodeau)\
**Post date:** [December 1, 2021, 1:40pm UTC](https://forum.mothur.org/t/cluster-split-issue-again-sorry/21196/4 "2021-12-01T13:40:12Z")

</div>

Alright, it ran through.

Took 17 hours. Now analyzing my run, I hope my positive controls are ok. for those that are interested, here is how I did it:

DNA extraction: lysis buffer, beads beating, heat, and phenol/chloro lots of DNA but I also have lots of Chimera probably due to DNA fragmentation, I will work on a less brutal extraction in the coming year)  
region: V4, Carporaso primers, so almost full overlaping  
number of samples: around 700  
number of sequences after cleaning: 45 818 071  
number of uniques: 390 779  
numbers of OTU (cluster.split, cutoff at 0.03): 71 770  
error rate: e-4 to e-6 depending on the positive controls.  
samples: chicken, ileal and caecal, different experiments done in our level 2 animal facility with one parameter in common that I want to check the impact for.

Batch file:  
set.current(processors=32)

set.logfile(name=megacampy\_logFile\_clustersplit)

make.contigs(file=megacampy.files, oligos=primers.oligo.txt, checkorient=t, pdiffs=0, maxee=2,deltaq=6)  
summary.seqs(fasta=current)

screen.seqs(fasta=current, group=current, summary=current, maxambig=0, maxhomop=70)  
count.groups(group=current)  
summary.seqs(fasta=current)

unique.seqs(fasta=current)  
count.seqs(name=current, group=current)  
summary.seqs(fasta=current, count=current)

align.seqs(fasta=current, reference=silva.nr\_v132.pcr.align, flip=t)  
summary.seqs(fasta=current, count=current)  
count.groups(group=current)

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

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

pre.cluster(fasta=current, count=current, diffs=4)  
count.groups(count=current)  
summary.seqs(fasta=current, count=current)

chimera.vsearch(fasta=current, count=current, dereplicate=t)  
remove.seqs(fasta=current, accnos=current)  
count.groups(count=current)  
summary.seqs(fasta=current, count=current)

classify.seqs(fasta=current, count=current, iters=1000, reference=silva.nr\_v138.align, taxonomy=silva.nr\_v138.tax, cutoff=80)

remove.lineage(fasta=current, count=current, taxonomy=current, taxon=Chloroplast-Mitochondria-unknown-Eukaryota)  
summary.tax(taxonomy=current, count=current)  
summary.seqs(fasta=current, count=current)  
count.groups(count=current)

#dist.seqs(fasta=current,cutoff=0.04)

#cluster.split(column=current, count=current, taxonomy=current, splitmethod=classify, taxlevel=6, delta=0, iters=500, cutoff=0.03)  
cluster.split(fasta=current, count=current, taxonomy=current, taxlevel=6, delta=0, iters=500, precision=1000, cutoff=0.03)

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

classify.otu(list=current, count=current, taxonomy=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Positif\_PCR\_sophie)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Zymo\_Ctrl\_PCR\_carvacrol)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=zymo\_extraction\_carvacrol)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Zymo\_community\_sophie)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Positif\_PCR2\_sophie)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=Commart\_probio)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

summary.seqs(fasta=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=megacampy.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count\_table)  
count.groups(count=current)

get.groups(count=current, fasta=current,taxonomy=current, groups=campy\_protease\_Zymo\_extraction)  
summary.seqs(fasta=current, count=current)  
seq.error(count=current, fasta=current, reference=zymostd\_ref.txt, aligned=F)

quit()

Kind regards and always use the latest version available…

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<div class="post-metadata">

**Author:** ![system](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/system/32/2_2.png) [@system](https://forum.mothur.org/u/system)\
**Post date:** [December 11, 2021, 1:41pm UTC](https://forum.mothur.org/t/cluster-split-issue-again-sorry/21196/5 "2021-12-11T13:41:08Z")

</div>

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