#create flowgram file from sff from 454 machine #sffinfo(sff=test.sff,flow=T) #trim and bin sequences in flowgram #trim.flows(flow=test.flow, oligos=test.oligos,bdiffs=0,pdiffs=1,processors=2) #denoise to remove sequencing errors, create fasta and qual files #shhh.flows(file=test.flow.files, processors=2) #bin by barcode, trim fasta files, remove low quality sequence #trim.seqs(fasta=test.shhh.fasta, name=test.shhh.names, oligos=test.oligos,flip=T,minlength=200,maxlength=500,maxambig=0,maxhomop=8,bdiffs=0,pdiffs=1,processors=2) #remove redundant sequences #unique.seqs(fasta=test.shhh.trim.fasta, name=test.shhh.trim.names) #align sequences to template 16S rRNA gene #align.seqs(fasta=test.shhh.trim.unique.fasta, reference=silva.bacteria/silva.bacteria.fasta, processors=2) #summarize results so far #summary.seqs(fasta=test.shhh.trim.unique.align, name=test.shhh.trim.names) #determine clear-span region #screen.seqs(fasta=test.shhh.trim.unique.align, name=test.shhh.trim.names, group=test.shhh.groups, end=6333, optimize=start, criteria=85, processors=2) #remove sequences and cut alignment to clear span region #filter.seqs(fasta=test.shhh.trim.unique.good.align, vertical=T, trump=., processors=2) #remove redundant sequences #unique.seqs(fasta=test.shhh.trim.unique.good.filter.fasta, name=test.shhh.trim.good.names) #pre cluster sequences #pre.cluster(fasta=test.shhh.trim.unique.good.filter.unique.fasta, name=test.shhh.trim.unique.good.filter.names, group=test.shhh.good.groups, diffs=2) #run ClimeraSlayer to identify potential chimeras #chimera.uchime(fasta=test.shhh.trim.unique.good.filter.unique.precluster.fasta, name=test.shhh.trim.unique.good.filter.unique.precluster.names, group=test.shhh.good.groups, processors=2) #remove chimeras identified by ChimeraSlayer #remove.seqs(accnos=test.shhh.trim.unique.good.filter.unique.precluster.uchime.accnos, fasta=test.shhh.trim.unique.good.filter.unique.precluster.fasta, name=test.shhh.trim.unique.good.filter.unique.precluster.names, group=test.shhh.good.groups)