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)
unique.seqs()
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 http://www.mothur.org/wiki/MiSeq_SOP#OTU-based_analysis 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!
NS