20180205_MiSeq_SOP_commands.md

Mothur commands used 20180205

Combining forward and reverse reads

make.contigs(file=stability.files, processors=2)

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

Remove sequence that are too long, or have ambigous bases

screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, maxambig=0, maxlength=275)

Repeat of above, but using the summary output as well

screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, maxambig=0, maxlength=275, summary=stability.trim.contigs.summary)

Obtaining what is in memory

get.current()

summary.seqs(fasta=current)

Creating unique sequences by clustering identical sequences, creates a names file

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

Counting sequences in the names file to create a count_table

count.seqs(name=stability.trim.contigs.good.names, group=stability.contigs.good.groups) Check that the output file has all the groups in various columns.

Summary of the dataset sofar, note this uses the fasta, groups and names files as well

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

Extracting the 16S rRNA V4 region from the silva.bacteria.fasta database

pcr.seqs(fasta=silva.bacteria.fasta, start=11894, end=25319, keepdots=F, processors=2)

Renaming the seqs.pcr output file

NOTE this command does not work in mothur on the biolinux machine. This needs to be done manually!!!

Nonetheless here is the command: rename.file(input=silva.bacteria.pcr.fasta, new=silva.v4.fasta)

Summary of the silva.v4.fasta file

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

Aligning the contigs against the silva v4 dataset

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

Summary of the aligned sequences

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

Removing sequences that do not align correctly to the V4 region, or that have too many ambigous bases

this duplicated the last sequence in the count_table file. the duplication was manually removed

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 after screening

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

or

summary.seqs(fasta=stability.trim.contigs.good.unique.good.align, count=stability.trim.contigs.good.good.count_table, processors=2)

Filtering the alignment from empty columns and removing overhangs from the alignment

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

Rerunning the unique.seqs command to adjust for the sequences being indentical

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

Running precluster to account for sequencing and pcr errors

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