README.md

Note

nf-core Candidate Pipeline: This pipeline is developed according to nf-core DSL2 standards and guidelines, and is being prepared for submission to the nf-core community. Repository development URL: https://github.com/JD2112/nanometaepigenomics

Introduction

nanometaepigenomics (candidate: nf-core/nanometaepigenomics) is a clinical-grade, reproducible bioinformatics pipeline designed for Oxford Nanopore Technologies (ONT) metagenomic sequencing. It couples high-accuracy long-read assembly and metagenome-assembled genome (MAG) recovery with native bacterial epigenetic profiling (5mC/5hmC methylation calling and motif identification). Crucially, the pipeline leverages Nanomotif to link unbinned mobile genetic elements (plasmids and phages identified by geNomad) back to host bacterial MAGs via shared restriction-modification methylation motifs.

Pipeline Steps Overview

1. Raw Read Preflight QC:
   • Sequencing read QC (NanoPlot).
2. Basecalling & Quality Preprocessing:
   • Optional GPU-accelerated basecalling & modified base calling with MM/ML tags (Dorado).
   • Adapter trimming (Porechop_ABI) and length/quality filtering (Filtlong).
3. 2-Stage Host Decontamination:
   • Stage 1: Human GRCh38 privacy screening and depletion (Minimap2 + Samtools).
   • Stage 2: Background food matrix / declared host depletion (Minimap2 + Mosdepth).
4. Metagenomic Assembly & MGE Prediction:
   • Long-read metagenome assembly (metaFlye).
   • Plasmid and viral element identification (geNomad).
5. Binning, QC & Taxonomy:
   • Read mapping to contigs with Minimap2 and depth calculation with MetaBAT2 (jgi_summarize_bam_contig_depths).
   • Metagenomic binning (MetaBAT2 or SemiBin2).
   • MAG quality and completeness assessment (CheckM2).
   • Taxonomic classification (GTDB-Tk).
6. Meta-epigenomics & Functional Annotation:
   • Modification pileup generation (Modkit).
   • De novo bacterial methylation motif discovery and plasmid-to-host genome association (Nanomotif).
   • Antimicrobial resistance profiling (AMRFinderPlus).
   • Virulence factor screening (ABRICATE with VFDB) and plasmid replicon typing (PlasmidFinder).
   • Rapid prokaryotic genome functional annotation (Bakta).
7. Quality Control & Reporting:
   • Audit manifest generation (*_run_manifest.json) and clinical summary table (*_clinical_report.tsv).
   • Summary report with MultiQC.

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
SAMPLE_1,/path/to/reads/SAMPLE_1.fastq.gz
SAMPLE_2,/path/to/reads/SAMPLE_2.fastq.gz

Each row represents an ONT run with long reads (gzipped FASTQ or BAM with MM/ML tags). If starting directly from Oxford Nanopore raw signal data (.pod5), you can point the pipeline to your POD5 folder via --pod5_dir.

Launch the pipeline:

nextflow run JD2112/nanometaepigenomics \
   -profile <docker/singularity/conda> \
   --input samplesheet.csv \
   --human_fasta /path/to/GRCh38.fa \
   --host_fasta /path/to/food_host.fa \
   --outdir results/

Warning

Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters; see docs.

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

Pipeline output

To see the results of an example test run with a full size dataset refer to the results tab on the nf-core website pipeline page. For more details about the output files and reports, please refer to the output documentation.

Credits

nf-core/nanometaepigenomics was originally written by Jyotirmoy Das (@JD2112).

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 on the Slack #nanometaepigenomics channel (you can join with this invite).

Citations

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

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.