OTUs

This step generates Operational Taxonomic Units (OTUs). The following steps are executed to extract OTUs.

  1. Merging paired-end read sequences
  2. Filtering merged sequences using quality scores
  3. Dereplicating merged sequences
  4. Clustering sequences
  5. Chimera removal
  6. 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

```