COMPARATIVE_ANNOTATION

COMPARATIVE_ANNOTATION#

This Nextflow script implements a workflow for comparative annotation of metagenome-assembled genomes (MAGs).

COMPARATIVE_ANNOTATION Workflow#

Overview#

This Nextflow script implements a comprehensive and flexible comparative genomic analysis workflow. It performs various analyses including pangenome analysis, functional annotation, and visualization of results, with the ability to skip steps based on existing results.

Key Features#

  • Dynamic execution based on existing results

  • Pangenome analysis using PPanGGOLiN

  • Functional annotations: KEGG (KofamScan), virulence factors (VFDB), antibiotic resistance (CARD), CAZymes (dbCAN)

  • Genome similarity analysis using Skani

  • Protein function prediction using eggNOG-mapper

  • Gene-trait association analysis using Scoary2

  • Flexible visualization for both gene presence/absence and gene count data

Inputs#

  • Quality-controlled genome assemblies (FASTAs)

  • Metadata file with sample information

Outputs#

  • Annotation results for each analysis tool

  • Visualization results including interactive plots and heatmaps

  • Separate visualizations for gene presence/absence and gene count data

Main Parameters#

Parameter

Description

Default Value

inputDir

Input directory for genome files

"${launchDir}/results/metagenome/<span style="color:#00B050">BIN_ASSESSMENT</span>/bins_quality_passed"

metadata

Path to metadata file

"${launchDir}/selected_metadata.csv"

metacol

Column number in metadata to use

Required (no default)

outdir

Output directory

"${launchDir}/results/metagenome/<span style="color:#7FBDFF">COMPARATIVE_ANNOTATION</span>/${current_time}"

cpus

Number of CPUs to use

40

pan_identity

PPanGGOLiN identity threshold

0.8

pan_coverage

PPanGGOLiN coverage threshold

0.8

module_completeness

KEGG module completeness threshold

0.5

kofamscan_eval

KEGG KO e-value threshold

0.00001

VFDB_identity

VFDB identity threshold

50

VFDB_coverage

VFDB coverage threshold

80

VFDB_e_value

VFDB e-value threshold

1e-10

CAZyme_hmm_eval

CAZyme HMM e-value threshold

1e-15

CAZyme_hmm_cov

CAZyme HMM coverage threshold

0.35

Workflow Structure#

The workflow dynamically determines which steps to run based on the existence of previous results:

  1. Genome Preparation: Selects and prepares input genomes based on metadata.

  2. Annotation:

    • Prokka for gene prediction (if not already run)

    • PPanGGOLiN for pangenome analysis (if not already run)

    • KofamScan for KEGG annotation

    • VFDB for virulence factor annotation

    • CARD for antibiotic resistance gene detection

    • dbCAN for CAZyme annotation

    • Skani for genome similarity analysis

    • eggNOG for protein function prediction

  3. Visualization:

    • Creates visualizations for both gene presence/absence and gene count data

    • Includes heatmaps and interactive plots for each annotation type

  4. Additional Analyses:

    • Scoary2 for gene-trait association analysis

    • Metadata summary creation

Key Processes and Their Outputs#

Process

Output

run_prokka

Prokka annotation files

run_ppanggolin

Pangenome analysis results

run_kofamscan_annotation

KO matrix

run_VFDB_annotation

VFDB annotation results (PA and count)

run_rgi_CARD_annotation

CARD annotation results (PA and count)

run_dbCAN_annotation

CAZyme annotation results (PA and count)

run_skani_annotation

Genome similarity matrix

run_eggNOG

eggNOG annotation results

Visualization Processes#

Each visualization process creates separate outputs for gene presence/absence (PA) and gene count data:

  • run_kofamscan_visualization

  • run_VFDB_visualization

  • run_rgi_CARD_visualization

  • run_dbCAN_visualization

  • run_skani_visualization

Usage#

nextflow run <span style="color:#7FBDFF">COMPARATIVE_ANNOTATION</span>_apptainer.nf --metadata [path_to_metadata] --metacol [metadata_column] [additional_options]

Requirements#

  • Nextflow

  • Apptainer/Singularity containers with required tools

  • Input genomes and metadata file

For detailed information on parameters and usage, run the script with the --help flag.

Notes#

  • The workflow uses Apptainer (formerly Singularity) containers for tool execution.

  • Results are organized in a structured output directory for easy navigation.

  • The script includes error checking for critical parameters and input files.

  • Visualization results are designed to be interactive and easily interpretable.

  • The workflow is flexible and can resume from partial runs, optimizing resource usage.

Output Files Details#

Check out the HTML Content!

COMPARATIVE_ANNOTATION result viewer.

Contents