Question about pre.cluster in miseq SOP

Hi there,

I am a new user of mothur and was reviewing the stability.batch file that comes with the tutorial dataset for the Miseq SOP. I was wondering why the pre.cluster command is implemented twice in the batch file as follows:

make.contigs(file=SLOAE_samples.files, processors=32)
screen.seqs(fasta=current, group=current, maxambig=0, optimize=maxlength, criteria=95, maxhomop=8)
count.seqs(name=current, group=current)
align.seqs(fasta=current, reference=silva.bacteria.fasta)
screen.seqs(fasta=current, count=current, optimize=start=end, criteria=95)
filter.seqs(fasta=current, vertical=T, trump=.)
pre.cluster(fasta=current, count=current, diffs=2)
unique.seqs(fasta=current, count=current)
pre.cluster(fasta=current, count=current, diffs=2)
chimera.uchime(fasta=current, count=current, dereplicate=t)

I have read the entire explanation for the Miseq SOP at and it appears that the pre.cluster command is only used once after filter.seqs. Am I missing something about the rationale for running this command twice?

Thank you for your time and your help!


That was a typo and it should only be used after running unique.seqs.

Hi Pat,

Sorry for the late reply but many thanks for the clarification.