Oct 31, 2025

Public workspaceProtocol for whole shotgun metagenomics pipeline for the study of stool human digestive microbiota

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvo9ox7v4o/v1
  • Victoria Meslier1,
  • Yani Ren1,
  • Mallia Geiger1,
  • Marine Gilles1,
  • Alexandre Famechon1,
  • Aymeric David1,
  • Christian Morabito1,
  • Benoit Quinquis1,
  • Mathieu Almeida1
  • 1Université Paris Saclay, INRAE MetaGenoPolis, 78350 Jouy-en-Josas, France
Victoria Meslier
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvo9ox7v4o/v1
Protocol Citation: Victoria Meslier, Yani Ren, Mallia Geiger, Marine Gilles, Alexandre Famechon, Aymeric David, Christian Morabito, Benoit Quinquis, Mathieu Almeida 2025. Protocol for whole shotgun metagenomics pipeline for the study of stool human digestive microbiota. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9ox7v4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 17, 2024
Last Modified: November 14, 2025
Protocol Integer ID: 115788
Keywords: ASAPCRN, metagenomics, human gut microbiota, dna extraction, shotgun sequencing, bioinformatics, human digestive microbiota this protocol, protocol for whole shotgun metagenomics pipeline, human digestive microbiota, digestive microbiota, whole shotgun metagenomics pipeline, metagenomic read mapping, human gut, microbial composition, functional potentials of the human gut, study of stool, whole dna extraction, procedures for whole dna extraction, stool
Funders Acknowledgements:
Agence Nationale de la Recherche
Grant ID: ANR-11-DPBS-0001
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
Abstract
This protocol describes the procedures for whole DNA extraction, shotgun high throughput sequencing, metagenomic read mapping and bioinformatical pre-processing to determine the microbial composition and functional potentials of the human gut digestive microbiota.
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Guidelines
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Materials
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Troubleshooting
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Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
Ensure proper sampling and samples conservation before proceeding to DNA extraction.
Wet lab section : DNA extraction and Shotgun sequencing
DNA isolation procedure, adapted from dx.doi.org/10.17504/protocols.io.dm6gpjm11gzp/v1
High throughput WGS sequencing as described in [1]
Dry lab section: Bioinformatical procedures
QC validation and read mapping was performed using the parametres described in the attached document using the 10.4M gut gene catalog and the 8.4M oral gene catalog.
Supplementary QC check was performed using CroCoDeEL tool https://github.com/metagenopolis/CroCoDeEL
Determination of the microbial composition of high quality samples using MSPminer [10]
Determination of the functional potentials of high quality samples using in-house pipeline as described in [15]
Protocol references
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7.         Amine Ghozlane, Florence Thirion, Florian Plaza Oñate et al. Accurate profiling of microbial communities for shotgun metagenomic sequencing with Meteor2, 05 March 2025, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-6122276/v1]
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