Mothur for 16S Analysis
Links
The MiSeq SOP by Pat Schloss (link)
Downlaod binary and datasets
# Download binary release wget "https://github.com/mothur/mothur/releases/download/v1.39.5/Mothur.linux_64_static.zip" unzip Mothur.linux_64_static.zip # Download datasets wget "https://mothur.org/w/images/d/d6/MiSeqSOPData.zip" wget "https://mothur.org/w/images/9/98/Silva.bacteria.zip" wget "https://mothur.org/w/images/5/59/Trainset9_032012.pds.zip" # Expand the downloaded datasets unzip MiSeqSOPData.zip unzip Silva.bacteria.zip unzip Trainset9_032012.pds.zip rm *.zip # Run mothur mothur
Mothur commands
Command make.file:
make.file(inputdir=MiSeq_SOP, type=fastq, prefix=stability)
Output:
Setting input directory to: MiSeq_SOP/ Output File Names: MiSeq_SOP/stability.files
Problem
The output file should look like:
F3D0 F3D0_S188_L001_R1_001.fastq F3D0_S188_L001_R2_001.fastq F3D141 F3D141_S207_L001_R1_001.fastq F3D141_S207_L001_R2_001.fastq F3D142 F3D142_S208_L001_R1_001.fastq F3D142_S208_L001_R2_001.fastq F3D143 F3D143_S209_L001_R1_001.fastq F3D143_S209_L001_R2_001.fastq ...
The issued command produces:
MiSeq MiSeq_SOP/F3D0_S188_L001_R1_001.fastq MiSeq_SOP/F3D0_S188_L001_R2_001.fastq MiSeq MiSeq_SOP/F3D141_S207_L001_R1_001.fastq MiSeq_SOP/F3D141_S207_L001_R2_001.fastq MiSeq MiSeq_SOP/F3D142_S208_L001_R1_001.fastq MiSeq_SOP/F3D142_S208_L001_R2_001.fastq MiSeq MiSeq_SOP/F3D143_S209_L001_R1_001.fastq MiSeq_SOP/F3D143_S209_L001_R2_001.fastq ...
Merge paired reads
From now on, Mothur is running from the MiSeq_SOP directory. Fusing the commands will require the make.contigs command:
make.contigs(file=mapping.files, processors=8)
Summary
summary.seqs(fasta=mapping.trim.contigs.fasta)
Using 4 processors. Start End NBases Ambigs Polymer NumSeqs Minimum: 1 248 248 0 3 1 2.5%-tile: 1 252 252 0 3 3810 25%-tile: 1 252 252 0 4 38091 Median: 1 252 252 0 4 76181 75%-tile: 1 253 253 0 5 114271 97.5%-tile: 1 253 253 6 6 148552 Maximum: 1 502 502 249 243 152360 Mean: 1 252.811 252.811 0.70063 4.44854 # of Seqs: 152360 Output File Names: mapping.trim.contigs.summary