How to analyze 16S rRNA amplicon sequencing data with QIIME2 from raw reads to diversity metrics

Question

I have paired-end 16S V4 amplicon sequencing data (Illumina MiSeq, 250 bp PE reads) from 20 gut microbiome samples. I want to identify taxa, calculate alpha/beta diversity, and find differentially abundant taxa between two groups. What is the complete QIIME2 workflow?

Admin · Accepted Answer

Here is the complete QIIME2 workflow for paired-end 16S data:

**1. Import reads**
```bash
qiime tools import 
  --type 'SampleData[PairedEndSequencesWithQuality]' 
  --input-path manifest.csv 
  --input-format PairedEndFastqManifestPhred33V2 
  --output-path demux.qza
```

**2. Denoise with DADA2** (trim based on quality plots)
```bash
qiime dada2 denoise-paired 
  --i-demultiplexed-seqs demux.qza 
  --p-trim-left-f 0 --p-trim-left-r 0 
  --p-trunc-len-f 230 --p-trunc-len-r 220 
  --p-n-threads 8 
  --o-table table.qza 
  --o-representative-sequences rep-seqs.qza 
  --o-denoising-stats denoising-stats.qza
```

**3. Classify taxonomy (against SILVA 138)**
```bash
qiime feature-classifier classify-sklearn 
  --i-classifier silva-138-99-nb-classifier.qza 
  --i-reads rep-seqs.qza 
  --p-n-jobs 4 
  --o-classification taxonomy.qza
```

**4. Build phylogenetic tree**
```bash
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
```

**5. Diversity analysis**
```bash
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 diversity-results
```

**6. Differential abundance with ANCOM-BC**
```bash
qiime composition ancombc 
  --i-table table.qza 
  --m-metadata-file metadata.tsv 
  --p-formula 'group' 
  --o-differentials differentials.qza
```

Download the SILVA 138 classifier from: `https://data.qiime2.org/2024.2/common/silva-138-99-nb-classifier.qza`