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Profiling the Athletes’ Gut Microbiome: A Critical Methodological Perspective on 16S Metabarcoding and Shotgun Metagenomics
by
, , and
Junior Carlone
1,2,3,*
,
Ághata Cardoso da Silva Ribeiro
3,4, 
Attilio Parisi
2
,
Saverio Giampaoli
2 and
Alessio Fasano
3,5,6
1
Department of Neurosciences, Biomedicine and Movement, University of Verona, 37134 Verona, Italy
2
Department of Movement, Human and Health Sciences, University of Rome “Foro Italico”, 00135 Rome, Italy
3
Division of Pediatric Gastroenterology and Nutrition, Mass General for Children and Harvard Medical School, Boston, MA 02114, USA
4
Laboratório Alerta, Departamento de Medicina Interna, Divisão de Doenças Infecciosas, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo 04021-001, SP, Brazil
5
Department of Nutrition, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA
6
European Biomedical Research Institute of Salerno, 84121 Salerno, Italy
*
Author to whom correspondence should be addressed.
Biology 2026, 15(8), 600; https://doi.org/10.3390/biology15080600
Submission received: 18 March 2026
/
Revised: 7 April 2026
/
Accepted: 8 April 2026
/
Published: 10 April 2026
(This article belongs to the Special Issue The Role of Gut Microbiota in Human Metabolism and Disease)
Simple Summary
The technology used to investigate the gut microbiota of athletes determines not only the precision of the results but also the biological conclusions that can be drawn. This review compares two of the most widely used sequencing methods: a targeted approach that analyses a specific region of bacterial genetic material to identify which microorganisms are present and a broader approach that sequences the entire genetic material of a sample, providing information not only on microbial identity but also on the functional activity of those microorganisms within the host. Each approach offers distinct advantages and limitations in terms of cost, taxonomic resolution, and the biological insights it can provide. For researchers working with elite athletes, selecting an inappropriate sequencing method may result in the loss of critical information regarding microorganisms that directly influence performance. Practical, evidence-based guidance is therefore provided to help sports scientists select the most appropriate sequencing strategy for their research objectives, ultimately contributing to the development of personalized nutritional and recovery interventions for athletes.
Abstract
The growing interest in the role of the gut microbiome in athletic performance has led to the application of various sequencing technologies in this field. This review critically examines the sequencing methodologies used in microbiome studies on physical performance and sport, comparing their advantages, limitations, and applicability. In particular, the focus is on 16S metabarcoding and shotgun metagenomics, evaluating how these methodological approaches influence the interpretation of results in sports contexts. Close attention is directed toward technical challenges, methodological biases, and future perspectives, including emerging technologies and multi-omics approaches. This review aims to bridge the gap between methodological rigor and sports-specific applicability, providing evidence-based methodological guidance to support researchers in designing robust athlete microbiome studies and translating sequencing-derived findings into concrete applications for performance and sports health.
