WMS_FUNCTION

WMS_FUNCTION#

This Nextflow script implements a workflow for functional analysis of whole metagenome sequencing data using HUMAnN3.

Workflow Execution#

WMS_FUNCTION execution#
# Activate metafun conda environment
conda activate metafun
# Move to metafun directory where you cloned metafun from GitHub
(metafun) nextflow run nf_scripts/<span style="color:#7030A0">WMS_FUNCTION</span>_apptainer.nf --inputDir ${inputDir} --metadata ${metadata} --sampleIDcolumn ${column_number} --analysiscolumn ${column_number}

Workflow Overview#

The workflow performs the following steps:

  1. HUMAnN3 analysis on individual samples

  2. Parsing and normalization of HUMAnN3 outputs

  3. Functional analysis and visualization based on metadata

Result of this workflow provides functional profiles of metagenomic samples, which can be used for downstream comparative analyses.

Inputs and Outputs#

Inputs: Quality-controlled paired-end metagenomic reads (output from RAWREAD_QC workflow), metadata file. Outputs: HUMAnN3 results, normalized pathway abundance tables, functional analysis visualizations.

Default output directory is ${launchDir}/results/metagenome/<span style="color:#7030A0">WMS_FUNCTION</span>.

Process

InputDir

OutputDir

Note

humann3_run

${params.inputDir}

${params.outdir}/humann3

HUMAnN3 analysis results for each sample

humann3_parsing

Output from humann3_run

${params.outdir}/humann3_combined

Combined and normalized HUMAnN3 results

function_analysis

Output from humann3_parsing

${params.outdir}

Functional analysis visualizations

Parameters in WMS_FUNCTION Nextflow Script#

Parameter

Description

Default Value

Note

db_baseDir

Base directory for databases

/opt/database

Mounted path in apptainer environment

scripts_baseDir

Base directory for scripts

/scratch/tools/microbiome_analysis/scripts

Mounted path in apptainer environment

inputDir

Input directory containing filtered reads

"${launchDir}/results/metagenome/<span style="color:#FF0000">RAWREAD_QC</span>/read_filtered"

Output from RAWREAD_QC workflow

outdir

Output directory

"${launchDir}/results/metagenome/<span style="color:#7030A0">WMS_FUNCTION</span>"

metadata

Path to metadata file

"meta.csv"

sampleIDcolumn

Column number for sample IDs in metadata

1

analysiscolumn

Column number for analysis grouping

0

cpus

Number of CPUs to use

36

Descriptions of Processes in WMS_FUNCTION Workflow#

  1. HUMAnN3 Run: Executes HUMAnN3 on each sample’s paired-end reads.

  2. HUMAnN3 Parsing: Combines and normalizes the HUMAnN3 outputs from all samples.

  3. Function Analysis: Performs statistical analysis and creates visualizations based on the normalized HUMAnN3 results and metadata.

Tools Used in WMS_FUNCTION#

Tool

Purpose

Version

Default parameters

Parameters that can be selected

HUMAnN3

Functional profiling of microbial communities

3.0.0

--search-mode uniref90, --pathways metacyc

--threads ${task.cpus}

R

Statistical analysis and visualization

4.3.2

N/A

N/A

Usage Notes#

  • The input directory should contain paired-end read files processed by the RAWREAD_QC workflow.

  • Metadata file should be in CSV format with at least two columns: sample IDs and grouping information for analysis.

  • The script checks for the existence and non-emptiness of the input directory before proceeding.

  • HUMAnN3 databases (ChocoPhlAn and UniRef90) should be properly set up in the specified database directory.

Output file details and examples#

The workflow generates the following outputs in the specified outdir:

  • HUMAnN3 output files for each sample (gene families, pathway abundances, pathway coverages)

  • Combined and normalized pathway abundance tables

  • Functional analysis visualizations (e.g., heatmaps, PCA plots)

  • Statistical analysis results based on the metadata groupings

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