# Precluster command running for more than 3 days ( ITS dataset, unite v.10). Complete ITS workflow

**URL:** <https://forum.mothur.org/t/precluster-command-running-for-more-than-3-days-its-dataset-unite-v-10-complete-its-workflow/22462>\
**Category:** Commands in mothur\
**Created:** [November 5, 2026, 1:52pm UTC](https://forum.mothur.org/t/precluster-command-running-for-more-than-3-days-its-dataset-unite-v-10-complete-its-workflow/22462 "2026-11-05T13:52:19Z")\
**Posts on this page:** 1\
**Page:** 1

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**Author:** ![samvarthini](https://avatars.discourse-cdn.com/v4/letter/s/77aa72/32.png) [@samvarthini](https://forum.mothur.org/u/samvarthini)\
**Post date:** [November 5, 2026, 1:52pm UTC](https://forum.mothur.org/t/precluster-command-running-for-more-than-3-days-its-dataset-unite-v-10-complete-its-workflow/22462/1 "2026-11-05T13:52:19Z")

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Dear mothur team,

I’m analysing fungal ITS amplicon sequences (paired-end illumina fastq) with mothur using unite databse v.10. I followed the miseq SOP and my run has now been at the precluster step for more than 3 days without finishing.

The commands I have run so far are:

mothur \> make.file(inputdir=., type=fastq, prefix=stability)

mothur\> make.contigs(file=stability.files)

mothur\> summary.seqs(fasta=stability.trim.comtigs.fasta. count=stability.contigs.count\_table)

mothur\> screen.seqs(fasta=stability.trim.contigs.fasta, count=stability.contigs.count\_table, maxambig=0, maxlength=275, maxhomop=8)

mothur\> get.current()

mothur\> summary.seqs(fasta=stability.trim.contigs.good.fasta, count=stability.contigs.count\_table)

mothur\> summary.seqs(fasta=current, count=current)

mothur\> summary.seqs(count=current)

mothur\> unique.seqs(fasta=stability.trim.contigs.good.fasta, count=stability.contigs.count\_table)

mothur\>pre.cluster(fasta=stability.trim.contigs.good.unique.fasta, count=stability.contigs.good.count\_table, diffs=2)

My system has 8 processors and 16GB of RAM and uses Windows 11

I would be grateful if you could help me with two things:

Why is pre.cluster taking so long (more than 3 days) in my case?

What is the complete recommended mothur workflow for fungal ITS sequence data, from raw fastq files to the final step?

Thank you very much for your time and help.

Best regards,

Samvarthini K A