LowBioPipe

A Nextflow wrapper for statistical analysis of low biomass microbiome data

Overview

LowBioPipe is a Nextflow-based computational framework that serves as a wrapper for processing outputs from the nf-core/taxprofiler pipeline, specifically designed for downstream analysis of low-biomass microbiome samples. In addition to facilitating data processing, LowBioPipe implements comprehensive statistical analyses, data-mining, and data visualization techniques, including contaminant filtering, multi-rank taxonomic abundance profiling, and diversity metrics with graphical representations.

Pipeline Summary

1. Contaminant Filtering - Remove reads assigned to specified taxa from Kraken2 output
2. Recentrifuge Analysis - Refined taxonomic profiling with negative control integration
3. Abundance Tables - Generate taxa × samples matrices at species, genus, and phylum levels
4. Diversity Analysis - Alpha/beta diversity, PCoA ordination, clustering heatmaps, PERMANOVA

Requirements

• Nextflow ≥ 23.04.0
• Docker, Singularity, or Conda
• NCBI taxdump

Input: nf-core/taxprofiler Output

LowBioPipe requires output from nf-core/taxprofiler with Kraken2 per-read classifications:

nextflow run nf-core/taxprofiler \
    -profile docker \
    --input samplesheet.csv \
    --databases databases.csv \
    --outdir taxprofiler_results \
    --run_kraken2 \
    --kraken2_save_readclassifications

The --kraken2_save_readclassifications flag is required.

Installation

Clone the repository and run the installation script:

git clone https://github.com/Physics4MedicineLab/LowBioPipe.git
cd LowBioPipe
./install.sh

Conda Environment

Alternatively, create a conda environment with all dependencies:

conda env create -f environment.yml
conda activate lowbiopipe

Usage

nextflow run main.nf \
    --taxprofiler_results /path/to/taxprofiler/output \
    --kraken2_db_name <database_name> \
    --samples S1,S2,S3,S4 \
    --controls BLANK1,BLANK2 \
    -profile docker

For PERMANOVA statistical testing, provide a groups file:

nextflow run main.nf \
    --taxprofiler_results /path/to/taxprofiler/output \
    --kraken2_db_name <database_name> \
    --samples S1,S2,S3,S4 \
    --controls BLANK1,BLANK2 \
    --groups_file groups.tsv \
    -profile docker

Parameters

Required

Parameter	Description
--taxprofiler_results	Path to taxprofiler output directory
--kraken2_db_name	Kraken2 database name used in taxprofiler

Sample Configuration

Parameter	Description	Default
--samples	Sample IDs (comma-separated)	[]
--controls	Negative control IDs (comma-separated)	[]
--groups_file	Sample-to-group mapping for PERMANOVA	null

Reference Data

Parameter	Description	Default
--taxdump	NCBI taxdump directory	data/taxdump
--exclude_taxa	Contaminant TaxID file	config/contaminants_example.txt

Contaminant Filtering

Parameter	Description	Default
--filter_include_ancestors	Also filter ancestor taxa (walking up to root)	false
--filter_include_descendants	Also filter descendant taxa (walking down tree)	false

Recentrifuge

Parameter	Description	Default
--recentrifuge_min_score	Minimum score threshold	10

Abundance Tables

Parameter	Description	Default
--abundance_ranks	Taxonomic ranks to generate	['species','genus','phylum']
--abundance_aggregate	Aggregate taxa to specified rank	true
--abundance_min_count	Minimum total count threshold	1
--abundance_min_samples	Minimum samples threshold	2
--abundance_relative	Generate relative abundance tables	true
--abundance_keep_unranked	Keep taxa without resolvable rank	false

Output

Parameter	Description	Default
--outdir	Output directory	results

Output

results/
├── filtered_reads/          # Filtered Kraken2 classifications
├── recentrifuge/            # Recentrifuge reports (.html, .xlsx)
├── abundance/               # Count matrices and metadata
│   ├── *_counts_*.tsv
│   ├── *_relative_*.tsv
│   └── *_taxa_metadata_*.tsv
└── diversity/
    └── {species,genus,phylum}/
        ├── alpha_metrics_*.tsv           # Alpha diversity (observed OTUs, Shannon, Simpson, Chao1)
        ├── alpha_*_boxplot_*.png         # Alpha diversity boxplots
        ├── alpha_*_violin_*.png          # Alpha diversity violin plots
        ├── beta_braycurtis_*.tsv         # Bray-Curtis distance matrix
        ├── beta_jaccard_*.tsv            # Jaccard distance matrix
        ├── beta_aitchison_*.tsv          # Aitchison distance matrix (CLR-Euclidean)
        ├── pcoa_braycurtis_coords_*.tsv  # PCoA coordinates (Bray-Curtis)
        ├── pcoa_jaccard_coords_*.tsv     # PCoA coordinates (Jaccard)
        ├── pcoa_aitchison_coords_*.tsv   # PCoA coordinates (Aitchison)
        ├── pcoa_braycurtis_*.png         # PCoA plot (Bray-Curtis)
        ├── pcoa_jaccard_*.png            # PCoA plot (Jaccard)
        ├── pcoa_aitchison_*.png          # PCoA plot (Aitchison)
        ├── heatmap_*.png                 # Hierarchical clustering heatmap
        ├── permanova_braycurtis_*.txt    # PERMANOVA results (Bray-Curtis)
        └── permanova_aitchison_*.txt     # PERMANOVA results (Aitchison)

Input File Formats

Contaminant file - One TaxID per line:

9606        # Homo sapiens
1751056     # Flavobacterium ammonificans

Groups file - Tab-separated, no header:

sample1    group_A
sample2    group_A
sample3    group_B
sample4    group_B

Citation

If you use LowBioPipe, please cite:

Please also cite the underlying tools:

• nf-core/taxprofiler: Stamouli et al. (2023) doi:10.1101/2023.10.20.563221
• Recentrifuge: Martí (2019) doi:10.1371/journal.pcbi.1006967
• Kraken2: Wood et al. (2019) doi:10.1186/s13059-019-1891-0

License

MIT License. See LICENSE for details.