OTUs
This step generates Operational Taxonomic Units (OTUs). The following steps are executed to extract OTUs.
- Merging paired-end read sequences
- Filtering merged sequences using quality scores
- Dereplicating merged sequences
- Clustering sequences
- Chimera removal
- Taxonomic classification
Merging paired-end read sequences
As the first step, we will merge forward and backward read sequences into one. The resultant sequences will be used for next steps.
qiime vsearch merge-pairs \
\
--i-demultiplexed-seqs paired-end-demux-trimmed.qza \
--o-merged-sequences demux-joined.qza --o-unmerged-sequences demux-unjoined.qza
Filtering merged sequences using quality scores
Now we will use merged pairs and apply quality filtering.
qiime quality-filter q-score \
\
–-i-demux demux-joined.qza \
--p-min-quality 20
–-o-filtered-sequences demux-joined-filtered.qza–-o-filter-stats demux-joined-filter-stats.qza
Dereplicating merged sequences
This step identify duplicate sequences and remove them. This step reduces final sequences for clustering.
qiime vsearch dereplicate-sequences \
--i-sequences demux-joined-filtered.qza \
--o-dereplicated-table derep-joined-filtered_table.qza \
--o-dereplicated-sequences derep-joined-filtered_seqs.qza
Clustering sequences
qiime vsearch cluster-features-de-novo \
--i-table derep-joined-filtered_table.qza \
--i-sequences derep-joined-filtered_seqs.qza \
--p-perc-identity .97 \
--o-clustered-table cluster-table.qza \
--o-clustered-sequences cluster-seqs.qza \
--p-threads 8
Chimera removal
qiime vsearch uchime-ref \
--i-table cluster-table.qza \
--i-sequences cluster-seqs.qza \
--i-reference-sequences silva-138.1-ssu-nr99-seqs-515f-806r.qza \
--p-threads 4 \
--o-chimeras chimeras.qza \
--o-nonchimeras non-chimera.qza \
--o-stats chimera-stats.qza
qiime feature-table filter-features \
--i-table cluster-table.qza \
--m-metadata-file non-chimera.qza \
--o-filtered-table table-nc.qza"
qiime feature-table filter-seqs \
--i-data cluster-seqs.qza \
--i-table table-nc.qza \
--o-filtered-data seqs-nc.qza
Taxonomic classification
qiime feature-classifier classify-sklearn \
--i-classifier silva_classifier.qza \
--i-reads seqs-nc.qza \
--o-classification seqs-nc-otus.qza
```