ASPIRE: Amplicon Sequencing Profiler for Investigating Respiratory Ecosystems

ASPIRE is a Nextflow DSL2 workflow for ASV generation, taxonomy assignment, decontamination, metadata-linked ASV summaries, ecological analyses, association analyses linking ASVs with Volatile Organic Compounds (VOCs), network/module analyses, and optional ASV-to-MAG linkage.

Full user guide · Workflow test · Issues and feature requests

Quick start

Use a 64-bit Linux system with Git and Mamba on PATH. The launcher creates its own Nextflow/Java controller and process environments. First-time setup needs internet access for packages and configured references. See installation for requirements.

git clone https://github.com/hallamlab/ASPIRE.git
cd ASPIRE

The control workflow runs full taxonomy → independent TECH/BIO prevalence tests → union removal → reference screening → combined ASV filtering → metadata tables. Only biological samples face the 5,000-read inclusion cutoff; nonzero controls are retained for testing. The mock uses 0.1% relative abundance in at least one retained biological sample plus 5% nonzero prevalence across biological samples, separately from decontam's score thresholds.

Run the workflow test

Run the bundled aspire-quickstart fixture: approximately 2.5 MB, 46 libraries and 218,000 read pairs. The wrapper builds the data, runs the full benchmarked workflow and executes its validator:

./examples/run_quickstart.sh --output "$PWD/aspire-quickstart" --threads 4

The separate aspire-cami-mock larger demonstration contains 50 synthetic patients and 179 libraries. Both use the same full validator.

Success ends with All mock-run checks passed. Open aspire-quickstart/results/summary/report/ASPIRE_run_report.html to review accounting, module outputs and execution logs. See remote report viewing if running over SSH.

Run your own study

cp asv_pipeline_nextflow.yml my_study.yml

Edit the input/output paths, references, sample metadata, assay settings and enabled modules in my_study.yml. Follow the study walkthrough and input guide; the complete template contains placeholders and study-specific examples.

./run_asv_pipeline.sh my_study.yml

Use the same command to resume. See the user guide for resources, configuration, outputs and troubleshooting.

Optional primer removal

Enable Cutadapt primer trimming to detect and remove paired primers before fastp. Set all four fixed fastp clipping values to zero when enabling this module. The primer audit records retained and discarded pairs, and a cohort check requires a single amplicon family before ASV construction.

Workflow

Vector SVG · PDF · Workflow and data-flow diagrams

For the complete workflow, configuration reference and interpretation, use the Read the Docs guide. Report bugs and request features through GitHub issues.

Cite ASPIRE

If you use ASPIRE, please cite:

McLaughlin, R. J., Chen, S., Nag, A., Noonan, A. J. C., Bartolomeu, C., Borden, S. A., Lam, S., Myers, R., & Hallam, S. J. (2026). ASPIRE: the Amplicon Sequencing Profiler for Investigating Respiratory Ecosystems. bioRxiv, version 2 (12 August 2026). DOI: 10.64898/2026.08.05.743000 · Read version 2.