Healthy human gut microbiome: Towards standardized research

Evgeniya Glazunova; Polina Molodtsova; Ilya Grabarnik; Alexander Kurnosov; Irina Bikaeva; German Shipulin; Olga Zlobovskaya; Evgeniya Glazunova; Polina Molodtsova; Ilya Grabarnik; Alexander Kurnosov; Irina Bikaeva; German Shipulin; Olga Zlobovskaya

doi:10.3934/microbiol.2025034

AIMS Microbiology

2025, Volume 11, Issue 4: 786-820. doi: 10.3934/microbiol.2025034

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Review

Healthy human gut microbiome: Towards standardized research

1.
Federal State Budgetary Institution “Centre for Strategic Planning and Management of Biomedical Health Risks” of the Federal Medical and Biological Agency, Russia
2.
Applied Genomics Laboratory, ITMO University, Saint Petersburg, 197101 Russia
3.
N.M. Emmanuel Institute of Biochemical Physics of the Russian Academy of Sciences, Moscow, 119334 Russia

Received: 18 July 2025 Revised: 01 October 2025 Accepted: 14 October 2025 Published: 04 November 2025

Objective
An increasing number of international researchers are focusing on the taxonomic composition of fecal microbiota and its correlation with disorders. Thousands of researchers compare conditionally healthy cohorts to those with specific diseases to identify potential markers. However, clinical application requires assessing the feasibility of synthesizing these findings and establishing reference intervals for normal gut flora, at least at higher taxonomic levels.
Design
This study involves a systematic review and meta-analysis of human gut microbiota research based on 16S rRNA gene next-generation sequencing (NGS). Relevant research was sourced following the PRISMA guidelines. Descriptive statistics, linear regression analysis by weighted least squares method, Mann-Whitney test, and Benjamini-Hochberg procedure adjustments were employed. The study has been registered with PROSPERO (CRD42023431467).
Results
Of the 4,346 studies initially identified, 86 publications involving 20,748 unique participants met the quality criteria and were included in the analysis of the impact of fecal sample preparation on taxonomic composition. The phylotype composition, in relation to preprocessing methods and cohort locations, are presented as relative abundances (%): Bacillota (median 49.5–59.6%), Bacteroidota (28.0–33.4%), Pseudomonadota (3.4–5.9%), Actinomycetota (2.3–3.7%), Verrucomicrobiota (0.5–1.0%), Fusobacteriota (maximum 4.6%), and Euryarchaeota (maximum 2.11%). The content of 27 key family-level representatives was also evaluated. The well-known hypothesis regarding the influence of the homogenization stage on taxonomic composition was examined using generalized results.
Conclusion
While supported by a strong theoretical basis and evidence from individual practical cases, none of the phyla showed a statistically significant association and consistent relationship with sample preparation or cohort location when generalizing across studies after the two exceptionally large cohorts exclusion, both originating from a single research group. These findings underscore the need for strict methodological standardization in microbiome studies. Key features of the 16S NGS process accounting for these results are outlined, along with proposed optimizations for microbiome research.
- human gastrointestinal microbiota,
- fecal microbiome,
- normal gut microbiome,
- sample homogenization,
- bead beating,
- 16S rRNA gene sequencing
Citation: Evgeniya Glazunova, Polina Molodtsova, Ilya Grabarnik, Alexander Kurnosov, Irina Bikaeva, German Shipulin, Olga Zlobovskaya. Healthy human gut microbiome: Towards standardized research[J]. AIMS Microbiology, 2025, 11(4): 786-820. doi: 10.3934/microbiol.2025034

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Abstract

Objective

An increasing number of international researchers are focusing on the taxonomic composition of fecal microbiota and its correlation with disorders. Thousands of researchers compare conditionally healthy cohorts to those with specific diseases to identify potential markers. However, clinical application requires assessing the feasibility of synthesizing these findings and establishing reference intervals for normal gut flora, at least at higher taxonomic levels.

Design

This study involves a systematic review and meta-analysis of human gut microbiota research based on 16S rRNA gene next-generation sequencing (NGS). Relevant research was sourced following the PRISMA guidelines. Descriptive statistics, linear regression analysis by weighted least squares method, Mann-Whitney test, and Benjamini-Hochberg procedure adjustments were employed. The study has been registered with PROSPERO (CRD42023431467).

Results

Of the 4,346 studies initially identified, 86 publications involving 20,748 unique participants met the quality criteria and were included in the analysis of the impact of fecal sample preparation on taxonomic composition. The phylotype composition, in relation to preprocessing methods and cohort locations, are presented as relative abundances (%): Bacillota (median 49.5–59.6%), Bacteroidota (28.0–33.4%), Pseudomonadota (3.4–5.9%), Actinomycetota (2.3–3.7%), Verrucomicrobiota (0.5–1.0%), Fusobacteriota (maximum 4.6%), and Euryarchaeota (maximum 2.11%). The content of 27 key family-level representatives was also evaluated. The well-known hypothesis regarding the influence of the homogenization stage on taxonomic composition was examined using generalized results.

Conclusion

While supported by a strong theoretical basis and evidence from individual practical cases, none of the phyla showed a statistically significant association and consistent relationship with sample preparation or cohort location when generalizing across studies after the two exceptionally large cohorts exclusion, both originating from a single research group. These findings underscore the need for strict methodological standardization in microbiome studies. Key features of the 16S NGS process accounting for these results are outlined, along with proposed optimizations for microbiome research.

Acknowledgments

This work was supported by the Centre for Strategic Planning and Management of Biomedical Health Risks of the Federal Medical Biological Agency of Russia through the provision of study personnel.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

Centre for Strategic Planning and Management of Biomedical Health Risks of Federal Medical Biological Agency, 119121 Moscow, Russia.
Evgeniya Glazunova, Polina Molodtsova, Ilya Grabarnik, Alexander Kurnosov, Irina Bikaeva, German Shipulin and Olga Zlobovskaya^#.
Evgeniya Glazunova, Olga Zlobovskaya—conceived the research question and designed the study. Evgeniya Glazunova, Olga Zlobovskaya, Irina Bikaeva—extracted and collected data from the original studies, and evaluated the quality of evidence. Ilya Grabarnik, Polina Molodtsova—conducted the statistical analysis. Evgeniya Glazunova, Olga Zlobovskaya—original draft preparation. Alexander Kurnosov—created the figures. Olga Zlobovskaya, Evgeniya Glazunova, Ilya Grabarnik, and Polina Molodtsova—edited the manuscript. Olga Zlobovskaya and German Shipulin—supervised the study.
All authors acknowledge full responsibility for the analyses and interpretation of the report. All authors have read and approved the final manuscript. All authors had full access to all the data in the study and had the final decision to submit for publication.

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