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mothur 16s code_Ning.txt
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mothur 16s code_Ning.txt
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# Go into” /lustre/haven/courses/EPP622-2018Fa/nduan1”
$ ssh [email protected]
$ cd /lustre/haven/courses/EPP622-2018Fa/nduan1
# Create directories
$ mkdir nifa_project
$ cd nifa_project
$ mkdir analysis
# Open new window and upload raw_data to ACF
$ scp –r [email protected]:/lustre/haven/courses/EPP622- 2018Fa/nduan1/nifa_project
# Load and run fastqc function(FastQC v0.11.5)
$ module load fastqc
$ fastqc -t 2 -o . ../raw_data/
# Load and run Mothur function(Linux 64Bit Version, Mothur version=1.39.5, Release Date=3/20/2017)
$ module load Mothur
$ Mothur
# then go into the Mothur terminal and execute Mothur commands according to Miseq SOP
mothur >make.contigs(file=ning16s.file,oligos=oligo.file,processors=8,pdiffs=2)
mothur >summary.seqs(fasta=ning16s.trim.contigs.fasta,processors=16)
mothur >screen.seqs(fasta=ning16s.trim.contigs.fasta,group=ning16s.contigs.groups,summary=ning16s.trim.contigs.summary,processors=16,maxambig=0,maxlength=450)
mothur > summary.seqs(fasta=ning16s.trim.contigs.good.fasta,processors=16)
mothur > unique.seqs(fasta=ning16s.trim.contigs.good.fasta)
mothur > count.seqs(name=ning16s.trim.contigs.good.names,group=ning16s.contigs.good.groups)
mothur > summary.seqs(fasta=ning16s.trim.contigs.good.unique.fasta,count=ning16s.trim.contigs.good.count_table,processors=16)
mothur > align.seqs(fasta=ning16s.trim.contigs.good.unique.fasta,reference=silva_V3_V4,processors=16)
mothur > summary.seqs(fasta=ning16s.trim.contigs.good.unique.align,count=ning16s.trim.contigs.good.count_table,processors=16)
mothur > screen.seqs(fasta=ning16s.trim.contigs.good.unique.align,count=ning16s.trim.contigs.good.count_table, summary=ning16s.trim.contigs.good.unique.summary,start=200,end=17212,maxhomop=8,processors=16)
mothur > summary.seqs(fasta=current,count=current)
mothur > filter.seqs(fasta=ning16s.trim.contigs.good.unique.good.align, vertical=T, trump=.)
mothur > unique.seqs(fasta=ning16s.trim.contigs.good.unique.good.filter.fasta, count=ning16s.trim.contigs.good.good.count_table)
mothur > pre.cluster(fasta=ning16s.trim.contigs.good.unique.good.filter.unique.fasta, count=ning16s.trim.contigs.good.unique.good.filter.count_table,diffs=4,processors=16)
mothur > chimera.vsearch(fasta=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.count_table, dereplicate=t,processors=16)
mothur > remove.seqs(fasta=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.fasta, accnos=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos)
mothur > summary.seqs(fasta=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table)
mothur > classify.seqs(fasta=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table, reference=trainset9_032012.pds.fasta, taxonomy=trainset9_032012.pds.tax, cutoff=80)
mothur > remove.lineage(fasta=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table,taxonomy=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.taxonomy, taxon=Chloroplast;-Mitochondria;-unknown;-Archaea;-Eukaryota;)
mothur > summary.tax(taxonomy=current, count=current)
mothur > cluster.split(fasta=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta, count=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table, taxonomy=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.taxonomy,splitmethod=classify,taxlevel=4,cutoff=0.03,processors=16)
mothur > make.shared(list=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list, count=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table, label=0.03)
mothur > classify.otu(list=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list, count=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table,taxonomy=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pds.wang.pick.taxonomy, label=0.03)
mothur > count.groups(shared=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared)
mothur > sub.sample(shared=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared, size=19210)
mothur > dist.shared(shared=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared, calc=thetayc-jclass, subsample=19210)
mothur > tree.shared(phylip=ning16s.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.thetayc.0.03.lt.ave.dist)