qiime2-amplicon v1.0A

Immutable. This exact content never changes and is served at /api/v1/blob/c21340281064d339.

---
name: qiime2-amplicon
description: Processes paired-end 16S amplicon reads into QIIME 2 ASVs and taxonomy with retained artifact provenance. Validates paired FASTQ manifests, primer orientation, post-trimming read overlap, sample metadata, classifier compatibility, and per-sample read retention.
license: MIT
compatibility: Python 3.10+ for the standard-library validation helper; QIIME 2 2026.7 distribution with cutadapt, dada2, feature-table, feature-classifier and taxa plugins for execution. Install through the official conda/container distribution, not PyPI. Requires a matching trained classifier and network for installation/reference downloads.
metadata:
  version: "1.0"
  skill-author: K-Dense Inc.
  upstream-version: "2026.7"
---

# QIIME 2 paired-end 16S amplicons

Use for a demultiplexed, paired-end 16S assay with known primers, quality encoding, expected insert
length, and sample metadata. This bounded workflow imports reads, removes 5′ primers, denoises with
DADA2, classifies ASVs using an explicitly supplied classifier, and retains `.qza`/`.qzv` provenance.
Do not treat read counts as absolute cell counts or taxonomy assignments as strain identification.

## Establish the assay before running

- Confirm Phred+33, read orientation, primer sequences **as sequenced** in forward/reverse reads,
  and whether primers have already been removed. The bundled runner requires primers still
  present at the 5′ ends; it anchors Cutadapt matching and discards untrimmed pairs. Already
  trimmed reads need a direct import → DADA2 workflow with that omission recorded in provenance.
- Choose truncation positions from actual per-base quality and error profiles. `trunc-f/r` are
  positions **after primer removal**. The expected maximum insert length also excludes primers.
  Require `trunc_f + trunc_r - maximum_insert_length >= 12`; use a margin for length variation.
  The check predicts geometrical overlap, not successful biological merging.
- Match classifier reference database, release, primer region, orientation and QIIME/scikit-learn
  compatibility. Record its source URL, database version and checksum. Do not automatically fetch
  an arbitrary “latest” classifier or reuse an incompatible serialized sklearn model. Load sklearn
  classifier artifacts only from trusted sources: QZA format validation does not make an
  untrusted serialized model safe.
- Include extraction blanks, PCR negatives, and a mock community where available. The runner
  rejects empty FASTQs; preserve empty-control IDs separately and report them rather than silently
  deleting control evidence. Assess contamination before ecological interpretation.

## Input files

Manifest is a **tab-separated** `PairedEndFastqManifestPhred33V2` file with exactly these headers:

```text
sample-id	forward-absolute-filepath	reverse-absolute-filepath
sample1	/data/sample1_R1.fastq.gz	/data/sample1_R2.fastq.gz
```

Use actual tab characters, absolute paths visible to the runtime, and one row per sample.
Do not reverse-complement R2 files. Sample metadata is a tab-separated file with first column
`sample-id`, unique IDs matching the manifest, and optional `#q2:types` annotation. Include
covariates and biological replicate IDs needed downstream.

## Execute

The helper lives at [scripts/amplicon_workflow.py](scripts/amplicon_workflow.py). First validate
without QIIME. These example primers and lengths are **illustrative**, not universal assay settings:

```bash
python scripts/amplicon_workflow.py validate \
  --manifest manifest.tsv --metadata sample-metadata.tsv \
  --primer-f GTGYCAGCMGCCGCGGTAA --primer-r GGACTACNVGGGTWTCTAAT \
  --trunc-f 220 --trunc-r 200 --amplicon-max 300
```

Then run in the QIIME 2 2026.7 environment with a compatible classifier:

```bash
python scripts/amplicon_workflow.py run \
  --manifest manifest.tsv --metadata sample-metadata.tsv \
  --primer-f GTGYCAGCMGCCGCGGTAA --primer-r GGACTACNVGGGTWTCTAAT \
  --trunc-f 220 --trunc-r 200 --amplicon-max 300 \
  --classifier region-classifier.qza --threads 4 --output run01
```

`run` executes immediately, writes only to a fresh output directory, and stops on a failing QIIME
command. It streams through every paired FASTQ record to catch mismatched IDs/order/counts,
checks sequence/quality consistency and sample alignment, and profiles the first 1,000 read pairs
for exact IUPAC primer matches. Low exact-match rates are warnings: Cutadapt allows mismatches,
but a low rate can also indicate incorrect orientation, adapters, or already-trimmed data.

The runner uses `--output-dir` for plugin methods with evolving output sets, preserving Cutadapt
statistics and DADA2 base-transition artifacts when supplied by the release. The 2026.7 table
summary also produces `feature-frequencies.qza` and `sample-frequencies.qza` beside `table.qzv`.
It records
`qiime-info.txt`, `commands.json`, `workflow.log`, input QC, classifier checksum and output artifact
checksums. It runs maximum-level QIIME artifact validation before reporting completion.
See [references/runtime-and-interpretation.md](references/runtime-and-interpretation.md) for the
release-pinned runtime, actual validation scope, restart handling and scientific interpretation.

## Inspect results before analysis

Open `trimmed.qzv`, `table.qzv`, and `taxa.qzv` in a local QIIME visualization environment or
[QIIME 2 View](https://view.qiime2.org/) as appropriate for the data. Examine quality/length profiles,
per-sample depth and dominant taxa. Retain original artifacts rather than replacing them with
CSV/BIOM exports: exports do not retain the original provenance graph.

`retention-qc.json` compares raw pairs with DADA2 input and non-chimeric reads, so trimming losses
remain visible. A <50% retained fraction is a review heuristic, not a universal rejection rule.
Inspect the individual stages in `stats/stats.tsv`: filtering loss suggests quality/expected-error
settings; merging loss suggests overlap or orientation; chimera loss warrants reviewing library
quality and parameters. Investigate missing/zero samples and control behavior before rarefaction,
diversity, or differential abundance. Those downstream analyses need a separate design decision;
this skill does not choose a rarefaction depth automatically.

## Primary references

- [Current installation entry point](https://library.qiime2.org/quickstart/qiime2) and
  [amplicon documentation](https://amplicon-docs.qiime2.org/en/latest/).
- [Import formats](https://amplicon-docs.qiime2.org/en/latest/how-to-guides/how-to-import/).
- [Cutadapt actions](https://amplicon-docs.qiime2.org/en/latest/references/plugins/cutadapt/) and
  [DADA2 actions](https://amplicon-docs.qiime2.org/en/latest/references/plugins/dada2/).
- [Classifier data resources](https://library.qiime2.org/data-resources).

The rolling documentation may describe a development release. Inspect `qiime info` and action
`--help` in the exact installed environment before adapting the pinned runner to a later release.