Q&A 4 How do you process raw sequencing data into a feature table using Mothur?
4.1 Explanation
Mothur is an open-source software package for analyzing 16S rRNA gene sequences. It supports raw data preprocessing, OTU clustering, and taxonomic assignment. Its workflow is particularly popular for microbial community studies and works well with single- or paired-end FASTQ data.
Mothur pipelines are usually run using command files or interactively within the Mothur console. Key steps include:
- Merging paired-end reads
- Quality filtering
- Aligning and clustering reads into OTUs
- Generating .shared (feature table) and .taxonomy files
4.2 Shell Code
# Launch Mothur
mothur
# Inside Mothur console (example workflow)
mothur > make.file(inputdir=./raw_data, type=fastq, prefix=stability)
mothur > make.contigs(file=stability.files, processors=8)
mothur > screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, maxambig=0, maxlength=275)
mothur > unique.seqs(fasta=stability.trim.contigs.good.fasta)
mothur > count.seqs(name=stability.trim.contigs.good.names, group=stability.contigs.good.groups)
mothur > align.seqs(fasta=stability.trim.contigs.good.unique.fasta, reference=silva.seed_v138.align)
mothur > screen.seqs(...)
mothur > pre.cluster(...)
mothur > chimera.vsearch(...)
mothur > remove.seqs(...)
mothur > cluster.split(...)
mothur > make.shared(...)
mothur > classify.otu(...)