Journal of medical microbiology2026; 75(7); 002176; doi: 10.1099/jmm.0.002176

Rapid bacterial community profiling of equine faecal, skin, milk and saliva samples using Oxford Nanopore long-read 16S rRNA amplicon sequencing.

Leng, J
·
Tait, C
·
Alsubaie, B
·
Van Vliet, AHM
·
Sells, P
·
La Ragione, RM
·
Proudman, C

Abstract: The composition of the equine gut microbiome is associated with many aspects of gastrointestinal, respiratory and musculoskeletal health that have been reported in the horse. Scientific studies exploring the microbiome non-intestinal ecological niches in or on horses are lacking. The clinical use of bacterial community profiling in horses is currently limited by cost and by slow analytical workflows. Most equine microbiome studies have relied on 16S rRNA amplicon sequencing of bacterial DNA, using high-throughput short-read sequencing technologies. This is often provided by an external service due to the cost of Illumina and other sequencers. Analysis of such sequencing files relies upon the researcher to have prior experience of coding-based programs. To explore the utility of Oxford Nanopore long-read sequencing in the analysis of microbiomes from several anatomical sites of the horse as a quicker and cheaper alternative to short-read sequencing. Bacterial DNA was extracted from horse (udder) skin swabs, saliva swabs, faecal samples and milk samples. Samples were prepared for Oxford Nanopore long-read sequencing and sequenced using a flow cell on the MinION Mk1D. Sequencing data were analysed using EPI2ME, along with extra analyses on exported taxa abundance data in R. Diversity measures and taxonomic relative abundance from phylum to family level were comparable to previously published equine studies that used Illumina sequencing. Sequencing data were acquired within 3 days costing around £30 per sample. Long-read sequencing gave accurate taxa assignment for two positive controls included at phylum, class, order and family levels of taxonomic classification. This work demonstrates that long-read technologies such as Oxford Nanopore MinION sequencing can provide a reliable, quick and cost-effective alternative to short-read Illumina sequencing when characterizing microbial communities from a range of anatomical locations on/in the horse.

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Publication Date: 2026-07-06 PubMed ID: 42406610PubMed Central: PMC13336655DOI: 10.1099/jmm.0.002176Google Scholar: Lookup

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Journal Article

Summary

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Overview

This study evaluates the use of Oxford Nanopore long-read 16S rRNA amplicon sequencing as a rapid and cost-effective method to profile bacterial communities in various equine sample types, including faecal, skin, milk, and saliva samples.
The research shows that long-read sequencing provides comparable results to traditional Illumina short-read sequencing but with faster turnaround and reduced costs, making it a promising tool for equine microbiome studies.

Introduction and Background

The equine gut microbiome has well-documented links to the health of horses, affecting gastrointestinal, respiratory, and musculoskeletal systems.
However, microbiome research in non-intestinal sites of horses—such as skin, milk, and saliva—is limited.
Traditional microbiome profiling mostly uses 16S rRNA gene sequencing with short-read technologies like Illumina, which are expensive and require external sequencing services and bioinformatics skills.
These cost and workflow factors limit the clinical and research applications of bacterial community profiling in horses.

Objectives

To assess the utility of Oxford Nanopore Technologies’ MinION long-read sequencing platform for rapid and affordable microbiome profiling from multiple anatomical sites in horses.
To compare sequencing results with those from previously published Illumina-based studies and evaluate accuracy.
To develop a sequencing and analysis workflow that reduces turnaround time and cost.

Methods

Bacterial DNA was extracted from four types of equine samples: udder skin swabs, saliva swabs, faecal samples, and milk samples.
The 16S rRNA gene was amplified and prepared for sequencing using Oxford Nanopore’s long-read technology.
Samples were sequenced on the MinION Mk1D platform using a flow cell.
Basecalling and taxonomic assignment were performed using the EPI2ME software platform, which is designed for Nanopore data and provides an accessible interface without requiring deep bioinformatics expertise.
Further analyses of taxa abundance and diversity metrics were conducted using R statistical software to compare results with prior Illumina data.
Positive control samples with known bacterial composition were included to assess classification accuracy.

Results

The Nanopore sequencing workflow yielded comprehensive bacterial community profiles within 3 days from sample preparation to data analysis.
Cost per sample was approximately £30, considerably lower than typical Illumina sequencing runs.
Measures of diversity (e.g., richness and evenness) and relative abundance of bacterial taxa at phylum, class, order, and family levels were consistent with published Illumina-based equine microbiome studies.
Positive control samples were correctly classified with high accuracy across multiple taxonomic levels, validating the reliability of the long-read sequencing data.
The use of long-read sequencing enabled better resolution of full-length 16S rRNA genes compared to short-read sequencing, potentially improving taxonomic assignment fidelity.

Discussion and Implications

Oxford Nanopore long-read 16S rRNA amplicon sequencing is demonstrated here to be a viable alternative to short-read technologies for equine microbiome profiling.
This method is faster and more cost-effective, potentially enabling broader adoption in veterinary research and clinical diagnostics.
Its portability (MinION device), lower cost, and simplified analysis pipeline reduce dependency on external sequencing providers and extensive bioinformatics expertise.
The ability to accurately profile microbial communities from various anatomical sites can help expand understanding of the equine microbiome beyond the gut alone.
Limitations include the need for continued validation and optimization to fully establish this technology across different sample types and complex microbial communities.

Conclusion

This study supports the use of Oxford Nanopore MinION sequencing as a reliable, rapid, and economical tool for detailed bacterial community analysis in horses.
Such advances have the potential to accelerate microbiome research and improve clinical interventions related to equine health.

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APA

Leng J, Tait C, Alsubaie B, Van Vliet AHM, Sells P, La Ragione RM, Proudman C. (2026). Rapid bacterial community profiling of equine faecal, skin, milk and saliva samples using Oxford Nanopore long-read 16S rRNA amplicon sequencing. J Med Microbiol, 75(7), 002176. https://doi.org/10.1099/jmm.0.002176

Publication

Journal of medical microbiology

ISSN: 1473-5644

NlmUniqueID: 0224131

Country: England

Language: English

Volume: 75

Issue: 7

PII: 002176

Researcher Affiliations

Leng, J

School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Daphne Jackson Road, Guildford GU2 7AL, UK.

Tait, C

School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Daphne Jackson Road, Guildford GU2 7AL, UK.

Alsubaie, B

School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Daphne Jackson Road, Guildford GU2 7AL, UK.
Department of Clinical Sciences, College of Veterinary Medicine, King Faisal University, Al-Hofuf 36388, Saudi Arabia.

Van Vliet, A H M

School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Daphne Jackson Road, Guildford GU2 7AL, UK.

Sells, P

Chasemore Farm, Orbital Veterinary Services, Bookham Road, Downside, Cobham KT11 3JT, UK.

La Ragione, R M

School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Daphne Jackson Road, Guildford GU2 7AL, UK.
School of Biosciences, Faculty of Health and Medical Sciences, Edward Jenner Building, University of Surrey, Guildford GU2 7XH, UK.

Proudman, C