Bio Microbiome Qiime2 Workflow

Denoise with DADA2

# DADA2 denoising (creates ASV table + representative sequences)
qiime dada2 denoise-paired \
    --i-demultiplexed-seqs demux.qza \
    --p-trunc-len-f 240 \
    --p-trunc-len-r 160 \
    --p-trim-left-f 0 \
    --p-trim-left-r 0 \
    --p-max-ee-f 2 \
    --p-max-ee-r 2 \
    --p-n-threads 0 \
    --o-table table.qza \
    --o-representative-sequences rep-seqs.qza \
    --o-denoising-stats denoising-stats.qza

# View denoising stats
qiime metadata tabulate \
    --m-input-file denoising-stats.qza \
    --o-visualization denoising-stats.qzv

Alternative: Deblur Denoising

# Quality filter first
qiime quality-filter q-score \
    --i-demux demux.qza \
    --o-filtered-sequences demux-filtered.qza \
    --o-filter-stats filter-stats.qza

# Deblur denoise
qiime deblur denoise-16S \
    --i-demultiplexed-seqs demux-filtered.qza \
    --p-trim-length 250 \
    --p-sample-stats \
    --o-representative-sequences rep-seqs-deblur.qza \
    --o-table table-deblur.qza \
    --o-stats deblur-stats.qza

Taxonomy Assignment

# Download pre-trained classifier (SILVA 138)
# wget https://data.qiime2.org/2024.5/common/silva-138-99-nb-classifier.qza

# Classify with sklearn naive Bayes
qiime feature-classifier classify-sklearn \
    --i-classifier silva-138-99-nb-classifier.qza \
    --i-reads rep-seqs.qza \
    --o-classification taxonomy.qza

# Visualize taxonomy
qiime metadata tabulate \
    --m-input-file taxonomy.qza \
    --o-visualization taxonomy.qzv

# Taxonomic barplot
qiime taxa barplot \
    --i-table table.qza \
    --i-taxonomy taxonomy.qza \
    --m-metadata-file metadata.tsv \
    --o-visualization taxa-barplot.qzv

Phylogenetic Tree

# Build phylogeny with MAFFT + FastTree
qiime phylogeny align-to-tree-mafft-fasttree \
    --i-sequences rep-seqs.qza \
    --o-alignment aligned-rep-seqs.qza \
    --o-masked-alignment masked-aligned-rep-seqs.qza \
    --o-tree unrooted-tree.qza \
    --o-rooted-tree rooted-tree.qza

Diversity Analysis

# Core metrics (alpha + beta diversity)
qiime diversity core-metrics-phylogenetic \
    --i-phylogeny rooted-tree.qza \
    --i-table table.qza \
    --p-sampling-depth 10000 \
    --m-metadata-file metadata.tsv \
    --output-dir core-metrics-results

# Alpha diversity significance
qiime diversity alpha-group-significance \
    --i-alpha-diversity core-metrics-results/shannon_vector.qza \
    --m-metadata-file metadata.tsv \
    --o-visualization shannon-significance.qzv

# Beta diversity significance (PERMANOVA)
qiime diversity beta-group-significance \
    --i-distance-matrix core-metrics-results/weighted_unifrac_distance_matrix.qza \
    --m-metadata-file metadata.tsv \
    --m-metadata-column Group \
    --p-method permanova \
    --o-visualization weighted-unifrac-permanova.qzv

Differential Abundance (ANCOM)

Goal: Identify taxa with significantly different abundances between groups using QIIME2's ANCOM implementation.

Approach: Collapse the feature table to genus level, add pseudocounts for log-ratio computation, and run ANCOM to test for differential abundance per taxon.

# Collapse to genus level
qiime taxa collapse \
    --i-table table.qza \
    --i-taxonomy taxonomy.qza \
    --p-level 6 \
    --o-collapsed-table table-l6.qza

# Add pseudocount and run ANCOM
qiime composition add-pseudocount \
    --i-table table-l6.qza \
    --o-composition-table comp-table-l6.qza

qiime composition ancom \
    --i-table comp-table-l6.qza \
    --m-metadata-file metadata.tsv \
    --m-metadata-column Group \
    --o-visualization ancom-l6.qzv

Export to R/Python

# Export feature table to BIOM
qiime tools export \
    --input-path table.qza \
    --output-path exported

# Convert BIOM to TSV
biom convert \
    -i exported/feature-table.biom \
    -o feature-table.tsv \
    --to-tsv

# Export taxonomy
qiime tools export \
    --input-path taxonomy.qza \
    --output-path exported

# Export tree
qiime tools export \
    --input-path rooted-tree.qza \
    --output-path exported

Manifest File Format

# manifest.tsv (tab-separated)
sample-id	forward-absolute-filepath	reverse-absolute-filepath
sample1	/path/to/sample1_R1.fastq.gz	/path/to/sample1_R2.fastq.gz
sample2	/path/to/sample2_R1.fastq.gz	/path/to/sample2_R2.fastq.gz

Metadata File Format

# metadata.tsv (tab-separated)
sample-id	Group	Timepoint
sample1	Treatment	Day0
sample2	Control	Day0
sample3	Treatment	Day7

Related Skills

• amplicon-processing - DADA2 R workflow alternative
• taxonomy-assignment - More database options
• diversity-analysis - phyloseq R alternative
• differential-abundance - ALDEx2/ANCOM-BC in R