Metagenomics pipeline using MetaPhlAn and HUMAnN

This pipeline can be used to obtain tax profiles, gene and pathway abundance tables and species-level genome bins from metagenomic reads. The input The MetaPhlAn and HUMAnN databases are in the current version downloaded as part of the pipeline and stored in the db folder (the first time you use the pipeline, it therefore takes a bit longer).

Introduction

metagenomicspipeline is a bioinformatics pipeline that processes shotgun metagenomics reads to obtain relative abundances and pathway abundances using BioBakery software.

1. Input check
2. Preprocessing
   • Read QC (FastQC)
   • Quality filtering and adapter trimming with fastp
   • Human read filtering: Bowtie2 to build an index and align reads with the human reference genome to filter out human reads and Samtools stats for read stats
   • Subsampling reads using seqkit
3. MetaPhlAn to obtain tax profiles from reads
4. HUMAnN to get gene and pathway abundance tables
5. Present a MultiQC report (MultiQC)

If you only want to use certain parts of the pipeline, you can use the flags --skip-processing, --skip-metaphlan, --skip-humann to skip certain subworkflows.

Usage

:::note If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data. :::

First, prepare a samplesheet with your input data that looks as follows:

samplesheet.csv:

sample,fastq_1,fastq_2
CONTROL_REP1,AEG588A1_S1_L002_R1_001.fastq.gz,AEG588A1_S1_L002_R2_001.fastq.gz

Each row represents a pair of fastq files. This pipeline does not accept single-end data.

Now, you can run the pipeline using:

nextflow run metagenomicspipeline \
   -profile <docker/singularity/.../institute> \
   --input samplesheet.csv \
   --outdir <OUTDIR>

Or run a test that uses the samplesheet_test.csv in the assets folder, using:

nextflow run metagenomicspipeline \
   -profile test,singularity \
   --outdir <OUTDIR>

If you are working on an HPC, there might be specific rules on how many jobs the pipeline can submit in a specific timeframe. I wrote a separate instruction for use on HPCs in the usage documentation

For more details and further functionality, please refer to the usage documentation and the parameter documentation.

Pipeline output

All output of the different parts of the pipeline are stored in subdirectories of the output directory. These directories are named after the tools that were used ('metaphlan', 'humann', etc.). Other important outputs are the multiqc report in the multiqc folder and the execution html report in the pipeline_info folder.

For more details on the pipeline output, please refer to the output documentation.

Credits

I used the nf-core template as much as possible and used the taxprofiler nf-core pipeline and Eduard's metagenomics pipeline as examples.

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines. For further information or help, don't hesitate to get in touch.

Citations

This pipeline uses bioBakery software, including MetaPhlAn and HUMAnN. Please cite their papers:

Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery

Francesco Beghini, Lauren J McIver, Aitor Blanco-Mìguez, Leonard Dubois, Francesco Asnicar, Sagun Maharjan, Ana Mailyan, Andrew Maltez Thomas,Paolo Manghi, Mireia Valles-Colomer, George Weingart, Yancong Zhang, Moreno Zolfo, Curtis Huttenhower, Eric A Franzosa, Nicola Segata

eLife 2021 10:e65088. doi: 10.7554/eLife.65088.

Extending and improving metagenomic taxonomic profiling with uncharacterized species using MetaPhlAn 4

Aitor Blanco-Miguez, Francesco Beghini, Fabio Cumbo, Lauren J. McIver, Kelsey N. Thompson, Moreno Zolfo, Paolo Manghi, Leonard Dubois, Kun D. Huang, Andrew Maltez Thomas, Gianmarco Piccinno, Elisa Piperni, Michal Punčochář, Mireia Valles-Colomer, Adrian Tett, Francesca Giordano, Richard Davies, Jonathan Wolf, Sarah E. Berry, Tim D. Spector, Eric A. Franzosa, Edoardo Pasolli, Francesco Asnicar, Curtis Huttenhower, Nicola Segata.

Nat Biotechnol. 2023. doi: 10.1038/s41587-023-01688-w.

If you use this metagenomicspipeline for your analysis, please cite it using the following doi: 10.5281/zenodo.10663326.

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.