WMS_TAXONOMY

WMS_TAXONOMY#

This Nextflow script implements a workflow for taxonomic analysis of whole metagenome sequencing data using Kraken2 and Bracken.

Workflow Execution#

WMS_TAXONOMY 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:#0846FA">WMS_TAXONOMY</span>_apptainer.nf --inputDir ${inputDir} --metadata ${metadata} --sampleIDcolumn ${column_number} --analysiscolumn ${column_number}

Workflow Overview#

The workflow performs the following steps:

  1. Kraken2 taxonomic classification

  2. Bracken abundance estimation

  3. Phyloseq object creation from Bracken outputs

  4. Statistical analysis and visualization based on metadata

Result of this workflow provides taxonomic 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: Kraken2 reports, Bracken abundance estimates, Phyloseq object, statistical analysis results and visualizations.

Default output directory is ${launchDir}/results/metagenome/<span style="color:#0846FA">WMS_TAXONOMY</span>.

Process

InputDir

OutputDir

Note

kraken2_run

${params.inputDir}

${params.outdir}/kraken2

Kraken2 classification results

bracken_run

Output from kraken2_run

${params.outdir}/bracken

Bracken abundance estimates

phyloseq_creation

Output from bracken_run

${params.outdir}/phyloseq

Phyloseq object creation

statistical_analysis

Phyloseq object

${params.outdir}/stats_analysis

Statistical analysis and visualizations

Parameters in WMS_TAXONOMY 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:#0846FA">WMS_TAXONOMY</span>"

metadata

Path to metadata file

Required input

sampleIDcolumn

Column number for sample IDs in metadata

1

analysiscolumn

Column number for analysis grouping

0

relab_filter

Relative abundance filter for Bracken results

0.000001

cpus

Number of CPUs to use

15

kraken_method

Kraken2 method (‘default’ or ‘memory-mapping’)

'default'

Descriptions of Processes in WMS_TAXONOMY Workflow#

  1. Kraken2 Run: Performs taxonomic classification on each sample’s paired-end reads using Kraken2.

  2. Bracken Run: Estimates abundances at the species level using Bracken, based on Kraken2 results.

  3. Phyloseq Creation: Creates a Phyloseq object from Bracken outputs and metadata for downstream analysis.

  4. Statistical Analysis: Conducts statistical analyses and creates visualizations based on the Phyloseq object and metadata.

Tools Used in WMS_TAXONOMY#

Tool

Purpose

Version

Default parameters

Parameters that can be selected

Kraken2

Taxonomic classification

2.1.2

--confidence 0.25, --paired

--memory-mapping

Bracken

Abundance estimation

2.7

-l S (species level)

-r ${read_length}

R (phyloseq)

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.

  • Kraken2 and Bracken databases should be properly set up in the specified database directory.

  • The kraken_method parameter allows for memory-mapping, which can improve performance on systems with sufficient RAM.

Output file details and examples#

The workflow generates the following outputs in the specified outdir:

  • Kraken2 classification reports for each sample

  • Bracken abundance estimates at the species level

  • Phyloseq object (RDS file) containing taxonomic profiles and metadata

  • Statistical analysis results and visualizations (e.g., alpha diversity plots, beta diversity ordinations, differential abundance analyses)

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