Using mothur: batch commands for processing sequencing data from Miseq

Inspired by & based on Mothur Miseq SOP

  1. Please try interactive mode before batch mode (to know what happens within each command)
  2. Please check numeric parameters within batch file before running it.

start from here

change the name of the file from stability.files to whatever suits your study

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, summary=stability.trim.contigs.summary, maxambig=0, maxlength=275)

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

summary.seqs(count=stability.trim.contigs.good.count_table)

###pcr.seqs(fasta=silva.bacteria.fasta, start=11894, end=25319, keepdots=F, processors=8) rename.file(input=silva.bacteria.pcr.fasta, new=silva.v4.fasta)###

summary.seqs(fasta=silva.v4.fasta)

align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.v4.fasta) summary.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table)

screen.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table, summary=stability.trim.contigs.good.unique.summary, start=1968, end=11550, maxhomop=8)

summary.seqs(fasta=current, count=current)

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) pre.cluster(fasta=stability.trim.contigs.good.unique.good.filter.unique.fasta, count=stability.trim.contigs.good.unique.good.filter.count_table, diffs=2)

chimera.vsearch(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table, dereplicate=t) remove.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, accnos=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos) summary.seqs(fasta=current, count=current)

classify.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, reference=trainset10_082014.pds.fasta, taxonomy=trainset10_082014.pds.tax, cutoff=80)

remove.lineage(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.taxonomy, taxon=Chloroplast-Mitochondria-unknown-Archaea-Eukaryota)

remove.groups(count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table, fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.taxonomy, groups=Mock)

dist.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.fasta, cutoff=0.01) cluster(column=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.dist, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.pick.count_table) cluster.split(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.taxonomy, splitmethod=classify, taxlevel=4, cutoff=0.01) make.shared(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.opti_mcc.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.pick.count_table, label=0.01) classify.otu(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.opti_mcc.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.taxonomy, label=0.01)

phylotype(taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.taxonomy) make.shared(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.tx.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.pick.count_table, label=1) classify.otu(list=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.tx.list, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.pick.count_table, taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.pick.taxonomy, label=1) dist.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.fasta, output=lt, processors=8) clearcut(phylip=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.phylip.dist) rename.file(taxonomy=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.opti_mcc.0.010.01.cons.taxonomy, shared=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.pick.opti_mcc.shared) count.groups(shared=stability.opti_mcc.shared) sub.sample(shared=stability.opti_mcc.shared, size=10000) rarefaction.single(shared=stability.opti_mcc.shared, calc=sobs, freq=100) summary.single(shared=stability.opti_mcc.shared, calc=nseqs-coverage-sobs-invsimpson-shannon, subsample=16000) heatmap.bin(shared=stability.opti_mcc.0.01.subsample.shared, scale=log2, numotu=50) dist.shared(shared=stability.opti_mcc.shared, calc=thetayc-jclass-braycurtis, subsample=5000) tree.shared(phylip=stability.opti_mcc.thetayc.0.01.lt.ave.dist) pcoa(phylip=stability.opti_mcc.thetayc.0.01.lt.ave.dist) nmds(phylip=stability.opti_mcc.thetayc.0.01.lt.ave.dist) nmds(phylip=stability.opti_mcc.thetayc.0.01.lt.ave.dist, mindim=3, maxdim=3) corr.axes(axes=stability.opti_mcc.thetayc.0.01.lt.ave.pcoa.axes, shared=stability.opti_mcc.0.01.subsample.shared, method=spearman, numaxes=3) get.communitytype(shared=stability.opti_mcc.0.01.subsample.shared) unifrac.unweighted(tree=current, count=current, distance=lt, processors=2, random=F, subsample=5000) unifrac.weighted(tree=stability.tre, count=stability.count_table, distance=lt, processors=2, random=F, subsample=5000)

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Jiayi Liu

Atypical Med student with “obsessive compulsory personality”. Interested in cancer research and bioinformatics. A heavy user of R. Crazy about any data that is logical, explicit, flat and sparse.

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