Hi everyone,

I have two paired-end fastq files from MiSeq platform with sequences of V3-V4 region of 16S rRNA and I want to cluster these samples. Following mothur SOP, I used next commands:

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

make.contigs(file=stability.files, processors=4)

summary.seqs(fasta=stability.trim.contigs.fasta)

screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, maxlength=570, minlength=400)

unique.seqs(fasta=stability.trim.contigs.good.fasta)

count.seqs(name=stability.trim.contigs.good.names, group=stability.contigs.good.groups)

align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.bacteria.fasta)

screen.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table, summary=stability.trim.contigs.good.unique.summary, start=6388, end=25316)

filter.seqs(fasta=stability.trim.contigs.good.unique.good.align, vertical=T, trump=.)

unique.seqs(fasta=stability.trim.contigs.good.unique.good.filter.fasta, count=stability.trim.contigs.good.good.count_table)

dist.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.fasta, cutoff=0.20)

I omitted some steps (chimera search, undesirables remove) consciously because they are not important for me now. But I am having problems with dist.seqs command - it is just producing several files with size > 40 Gb, until I am out of my hard drive space. What am I doing wrong?

Any help would be appreciated!