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Genotoul

Bioinfo

# MetagWS

A comprehensive workflow to analyze metagenomic data
using Illumina or PacBio HiFi reads

Jean MAINGUY*, Maïna VIENNE*, Joanna FOURQUET, Vincent DARBOT, Pierre MARTIN, Céline NOIROT, Philippe Ruiz, Adrien CASTINEL, Sylvie COMBES, Christine GASPIN, Denis MILAN, Cécile DONNADIEU, Carole IAMPIETRO, Olivier BOUCHEZ, Géraldine PASCAL and Claire HOEDE

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# Contents

* People involved

* The workflow features, steps and perspectives

* Support for biologists in metagenomics (& projects)
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GenOTOL
Bioinfo

People involved

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>Joanna Fourquet</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>Céline Noirot</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>Pierre Martin</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>Jean Mainguy</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>Théo Moser</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>Maria Bernard</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>Philippe Ruiz</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>Maïna Vienne</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>Vincent Darbot</figcaption></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

LABEX
ECOFECT
UNIVERSITÉ DE LYON

L'EUROPE S'ENGAGE
L'OCCITANIE AGIT

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<figure><img src="image_27.png" /></figure>

Projet cofinancé par le Fonds Européen de Développement Régional

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FRANCE
GENOMIQUE

SeqOccln

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anses

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OUEST

RESALAB

NOTRE PASSION, VOTRE SECURITE
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GenOToul
Bioinfo

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# metagWGS v2.5.0

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Freely available :

https://forge.inrae.fr/genotoul-bioinfo/metagwgs

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nextflow

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<figure><img src="image_37.png" /><figcaption># metagWGS v2.5.0</figcaption></figure>
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<figure><img src="image_39.png" /><figcaption># metagWGS v2.5.0</figcaption></figure>

Freely available :

https://forge.inrae.fr/genotoul-bioinfo/metagwgs
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GenOToul
Bioinfo

<figure><img src="image_41.png" /></figure>

# metagWGS v2.5.0

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Freely available :

https://forge.inrae.fr/genotoul-bioinfo/metagwgs

Binette already published in JOSS
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GenOToul
Bioinfo

# Binette

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

https://github.com/genotoul-bioinfo/Binette

Binette: a fast and accurate bin refinement tool to
construct high quality Metagenome Assembled
Genomes.

<figure><img src="image_46.png" /></figure>

Jean Mainguy Ⓟ 1,2,3 and Claire Hoede Ⓟ 1,2 ¶

1 Université de Toulouse, INRAE, BioinfOmics, GenoToul Bioinformatics facility, 31326, Castanet-Tolosan, France 2 Université de Toulouse, INRAE, UR 875 MIAT, 31326, Castanet-Tolosan, France 3 LABGeM, Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, Evry, France ¶ Corresponding author

DOI: 10.21105/joss.06782

Software

- Review ⚙

- Repository ⚙

- Archive ⚙

Editor: Arfon Smith ⚙️

Reviewers:

- @lskatz

- @beardymcjohnface

Submitted: 31 January 2024

Published: 05 October 2024

License

Authors of papers retain copyright
and release the work under a
Creative Commons Attribution 4.0
International License (CC BY 4.0).

Statement of need

Metagenomics enables the study of microbial communities and their individual members through shotgun sequencing. An essential phase of metagenomic analysis is the recovery of metagenome-assembled genomes (MAGs). MAGs serve as a gateway to additional analyses, including the exploration of organism-specific metabolic pathways, and form the basis for comprehensive large-scale metagenomic surveys (Acinas et al., 2021; Nayfach et al., 2019).

In a metagenomic analysis, sequence reads are first assembled into longer sequences called contigs. These contigs are then grouped into bins based on common characteristics in a process called binning to obtain MAGs. There are several tools that can be used to bin contigs into MAGs. These tools are based on various statistical and machine learning methods and use contig characteristics such as tetranucleotide frequencies, GC content and similar abundances across samples (Alneberg et al., 2014; Kang et al., 2019; Nissen et al., 2021).

The approach of applying multiple binning methods and combining them has proven useful to
obtain more and better quality MAGs from metagenomic datasets. This combination process is
called bin-refinement and several tools exist to perform such tasks, such as DASTool (Sieber et
al., 2018), MagScot (Rühlemann et al., 2022) and the bin-refinement module of the metaWRAP
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GenOToul
Bioinfo

Type of results

<figure><img src="image_48.png" /></figure>

<table><thead><tr><th>Level</th><th colspan="2">Reads</th><th>Genes in all contigs</th><th colspan="2">All contigs</th><th colspan="2">MAGS / bins</th></tr><tr><th>Tools</th><th>taxo</th><th>function</th><th>taxo</th><th>taxo</th><th>genes function</th><th>taxo</th><th>genes function</th></tr></thead><tbody><tr><td>MAG (nf-core)<br>Hybrid assembly<br>possible</td><td>YES</td><td>NO</td><td>NO</td><td>NO</td><td>NO</td><td>YES</td><td>YES (but not<br>rRNA and<br>tRNA)</td></tr><tr><td>Metawrap</td><td>YES</td><td>NO</td><td>NO</td><td>YES</td><td>NO</td><td>YES</td><td>YES</td></tr><tr><td>VEBA 2.0 (short and<br>long reads: ONT &<br>Pacbio)</td><td>NO</td><td>Only with<br>bins<br>found</td><td>NO</td><td>NO</td><td>NO</td><td>YES</td><td>YES</td></tr><tr><td>Atlas</td><td>NO</td><td>NO</td><td>NO</td><td>NO</td><td>YES</td><td>YES</td><td>YES</td></tr><tr><td>Anvi'o Metagenomic<br>workflow</td><td>YES</td><td>NO</td><td>NO</td><td>NO</td><td>YES</td><td>YES</td><td>YES</td></tr><tr><td>HiFi-MAGs-pipeline<br>(HiFi reads only,<br>binning only)</td><td>NO</td><td>NO</td><td>NO</td><td>NO</td><td>NO</td><td>YES</td><td>NO</td></tr><tr><td>metagWGS<br>(short_reads &amp; HiFi)</td><td>YES</td><td>NO</td><td>YES</td><td>YES</td><td>YES</td><td>YES</td><td>YES (via<br>contigs)</td></tr></tbody></table>
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GenOTOUl
Bioinfo

# Main perspectives in 2026

- Ability to analyze both short paired-end and single-end reads samples and PacBio samples (January 2026)

- Add the ability to analyze ONT data

- Overhaul of binning (new tools, new strategy, etc.)

- Improve modularity and disk occupation

- Internship 2026 with GenPhySE BeeGEES team (Julie Birgel & Thibault Leroy) & Philippe Bordron : construction of metabolic networks based on functions present in the Honey metagenome
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GenOTOUl
Bioinfo

# Support for biologists in metagenomics

- Documentation

- Training

- Scientific collaborations

Either we do the treatment or we supervise someone else to do it
(interns, PhD students, fixed-term contract workers, etc.)
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GenOToul
Bioinfo

# Support for biologists in metagenomics

## ☐ Documentation (Readthedocs and Sphinx)

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metagWGS

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Search docs

## CONTENTS:

<figure><img src="image_54.png" /></figure>

metagWGS: installation procedure

I. Install Nextflow

II. Install metagWGS

III. Install Singularity

IV. Download or build Singularity containers

metagWGS: Usage

metagWGS: Output

metagWGS: use case with big data test on genobioinfo cluster

Functional tests: Usage

<figure><img src="image_55.png" /></figure>

/ metagWGS: installation procedure

View page source

## metagWGS: installation procedure

### I. Install Nextflow

metagWGS is a Nextflow pipeline, you must install Nextflow to run it. Nextflow runs on most POSIX systems (Linux, Mac OSX etc). It can be installed by following the instructions [here]().

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#### Warning

Change your $PATH variable to run Nextflow in any directory.

For any issue during Nextflow installation and use, you can see [this tutorial]() and [this documentation]().

<figure><img src="image_57.png" /></figure>

#### Note

If you are on [genobioinfo cluster](), you don't need to install Nextflow.

You only need to know Nextflow version installed on genobioinfo.
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GenOTOUl
Bioinfo

# Support for biologists in metagenomics

<figure><img src="image_59.png" /></figure>

## Training

Home

About us

Resources

Training

Software development

Ask for

FAQ

<figure><img src="image_60.png" /></figure>

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GenOTOUl
Bioinfo

### ANALYSIS OF SHOTGUN METAGENOMIC DATA

#### ANALYSIS OF SHOTGUN METAGENOMIC DATA (05/05/2025 - 07/05/2025)

This training session is organized by the **Genotoul bioinfo platform**. This course is dedicated to **the analysis of prokaryotic shotgun metagenomic data from Illumina and Pacbio HiFi sequencing technology**.

After an overview of metagenomics and the biases and limitations of analyses, we will look at the main steps involved in analysing metagenomic data and launch independent tools on the genobioinfo cluster.

Learners will then test a workflow to automate processing on a test dataset [metagWGS](https://www.example.com).

On the third day, learners will choose which analysis strategy to start with according to their experimental design and launch the first stage of [metagWGS](https://www.example.com) on their own data.

By the end of the course, trainees will be familiar with the scope, advantages and limitations of shotgun sequencing data analysis and will have started the analysis on their own data.
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GenOTOUl
Bioinfo

# Support for biologists in metagenomics

□ Scientific collaborations in metagenomics

5 completed projects (biotechnologie, environment and antibiotic resistance)

Environmental Pollution 314 (2022) 120207

<figure><img src="image_63.png" /></figure>

ELSEVIER

Contents lists available at ScienceDirect

Environmental Pollution

journal homepage: www.elsevier.com/locate/envpol

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Université Fédérale

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Toulouse Midi-Pyrénées

## THÈSE

En vue de l'obtention du

Impact of environmental micropollutants and diet composition on the gut microbiota of wild european eels (Anguilla anguilla)☆

<figure><img src="image_66.png" /></figure>

**DOCTORAT DE L'UNIVERSITÉ DE TOULOU** Anthony Bertucci a, *, Claire Hoede
Karine Le Menach d, Hélène Budzi

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frontiers

Frontiers in Microbiology

Délivré par l'Institut National des Sciences Appliquées de Toulouse

EABX, INRAE, 50 Avenue de Verdun 33612, Cestas, France

b Université de Toulouse, INRAE, UR MIAT, PF GenoToul I

c Université Fédérale de Toulouse, INRAE, BioinfOmics, Ger

d Univ. Bordeaux, CNRS, EPOC, EPHE, UMR 5805, 33600

e Genome & Transcriptome - Plateforme GeT-PlaGe, INRAE

Présentée et soutenue par

<figure><img src="image_68.png" /></figure>

Check for updates

Louison DUMOND

Le 13 décembre 2021

Dégradation de la lignine par des consortia microbiens issus de termites : Enrichissement et caractérisation des communautés par des approches (bio-)chimiques et métagénomiques

### OPEN ACCESS

EDITED BY
Benoit Doublet,
INRAE, Université de Tours, France

REVIEWED BY
Minjae Kim,
University of Kentucky, United States
Cresten Mansfeldt,
University of Colorado Boulder, United States

*CORRESPONDENCE
Delphine Bibbal
Delphine.bibbal@envt.fr

RECEIVED 26 January 2024
ACCEPTED 11 March 2024
PUBLISHED 27 March 2024

CITATION
Naudin SA, Ferran AA, Imazaki PH,
Arpaillange N, Marcuzzo C, Vienne M,
Demmou S, Bousquet-Mélou A,

Development of an *in vitro* biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota

Sarah A. Naudin<fnref n="1" />, Aude A. Ferran<fnref n="1" />, Pedro Henrique Imazaki<fnref n="1" />,
Nathalie Arpaillange<fnref n="1" />, Camille Marcuzzo<fnref n="1" />, Mâina Vienne²,³,
Sofia Demmou⁴, Alain Bousquet-Mélou<fnref n="1" />,
Felipe Ramon-Portugal<fnref n="1" />, Marlene Z. Lacroix<fnref n="1" />, Claire Hoede²,³,
Maialen Barret⁴, Véronique Dupouy<fnref n="1" /> and Delphine Bibbal<fnref n="1" />*

TYPE Original Research
PUBLISHED 27 March 2024
DOI 10.3389/fmicb.2024.1377047

<footnote n="1">¹INTHERES, Université de Toulouse, INRAE, ENVT, Toulouse, France. ²Université de Toulouse, INRAE, BioinfOmics, GenoToul Bioinformatics Facility, Castanet-Tolosan, France. ³Université de Toulouse, INRAE, UR 875 MIAT, Castanet-Tolosan, France. ⁴Centre de Recherche sur la Biodiversité et l'Environnement, Université de Toulouse, CNRS, IRD, Toulouse INP, Université de Toulouse, Toulouse, France</footnote>
<figure><img src="image_69.png" /></figure>

GenOTOUl
Bioinfo

# Support for biologists in metagenomics

- ☐ Scientific collaborations

**13 active projects** (biotechnologie, **environment** and antibiotic resistance)

> 50% are environmental projects (sea water, waste water, biofilm, soil, tit faeces, honey ...)

6 PhD students supported for varying lengths of time and with different implications

<figure><img src="image_70.png" /><figcaption>Fabien Graziani</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>Philippe Ruiz</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>Théo Moser</figcaption></figure>

<figure><img src="image_73.png" /><figcaption>Nicolas Enjalbert-Courrech</figcaption></figure>
<figure><img src="image_74.png" /></figure>

GenOTOUl
Bioinfo

# Focus on the Panoramics project (ANR)

☐ Oriane Della-Negra, Rémi Servien, Kim Milferstedt, Jérôme Hamelin (INRAE, Univ. Montpellier, LBE, Narbonne); Christophe Klopp; Marie-Ange Teste (GeT-Biopuce, TBI)

☐ Metagenomic analysis and metatranscriptomic analysis of photogranules

Photogranules are composed of syntrophically interacting cyanobacteria and heterotrophic bacteria: cyanobacteria produce oxygen for heterotrophs, which in return provide carbon dioxide to cyanobacteria. Photogranules used in this study originated from a lab-scale photobioreactor fed with synthetic wastewater.
<figure><img src="image_75.png" /></figure>

GenOToul
Bioinfo

# Panoramics metagenome assembly

* 1 sample sequenced in Pacbio Revio HiFi to build the reference metagenome onto which RNASeq data has been mapped (25 samples)

* 1,8 Millions HiFi reads (length: ~10 000pb)

<table><thead><tr><th></th><th></th><th>Largest<br>contig<br>(Kbp)</th><th>Length<br>(Mbp)</th></tr></thead><tbody><tr><td>N50 (Kbp)</td><td>L50 (K)</td><td></td><td></td></tr><tr><td>407.7Kbp</td><td>0.2K</td><td>6427.6Kbp</td><td>350.0Mbp</td></tr></tbody></table>
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GenOTOL
Bioinfo

# Panoramics metagenome assembly

<figure><img src="image_77.png" /></figure>
<figure><img src="image_78.png" /></figure>

GenOTOL
Bioinfo

# We builds 25 High-quality MAGs

<figure><img src="image_79.png" /></figure>

They were
annotated with
Prokka, classified
using GTDB-Tk,
and functionally
annotated using
eggNOG-mapper

Created with MultiQC
<figure><img src="image_80.png" /></figure>

GenOTOUl
Bioinfo

# Focus on 25 high-quality MAGs

<figure><img src="image_81.png" /><figcaption>Fig 1. Phylogenetic tree, built from msa R-package v1.28 and phangorn v2.11.1, showing the placement of the 25 MAGs recovered from the oxygenic photogranules. Heatmaps show the module completeness obtained from eggNOG-mapper v2.1.9 annotation, associated with Nitrogen Metabolism (ko 00910) and Photosynthesis (ko 00195).</figcaption></figure>
<figure><img src="image_82.png" /></figure>

Thanks to

ANR Project Panoramics (grant number ANR-21-CE45-0036-0).

GENTYANE Platform UMR INRAE/UCA GDEC
Clermont-Ferrand

The Environmental Biotechnology and Biorefinery
Facility (Bio2E) of INRAE-LBE
(doi.org/10.15454/1.557234103446854E12) for
photogranule production.
Thank you for your attention

☐ Do you have questions ?

To ask to collaborate with us :

https://bioinfo.genotoul.fr/index.php/ask-for/project/
<figure><img src="image_83.png" /></figure>

GenOTOUl
Bioinfo

<figure><img src="image_84.png" /><figcaption>Cumulative length of
sequences by quality category</figcaption></figure>

Created with MultiQC