Animal genetics2026; 57(4); e70170; doi: 10.1002/age.70170

Approaches to Thoroughbred Horse Identification in Diverse Sample Types Using Multiplexed SNP-PCR Sequencing Panels.

Maniego, J
·
Harding, C
·
Hincks, P
·
Habershon-Butcher, J
·
Ryder, E

Abstract: The ability to determine the identity of equine samples would be highly advantageous in cases of ambiguous sample source, as individual-specific controls for gene editing detection, or forensic examination of suspect biological materials in potential doping cases. To this end, a SNP-PCR sequencing-based panel comprising 93 markers in one reaction was designed and investigated for use in Thoroughbred horses. Marker amplification performance and discriminating power were calculated against a population survey of 264 Thoroughbred whole blood samples from routine doping testing. The sensitivity of the SNP panel was tested on a dilution series and on a variety of matrices, including different types of blood specimen, hair and faeces. No genotyping errors due to allelic dropout were detected down to a starting concentration of 0.25 ng/μL in the dilution series, with both EDTA and lithium heparin anticoagulants performing comparably. Forensic 'crime scene' samples, such as blood on a blood tube label, also performed strongly, although others were more variable due to low DNA concentrations. A mock case of 10 'suspects' and one test blood sample was performed blind by the analyst who correctly identified the source of the sample.

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Publication Date: 2026-07-29 PubMed ID: 42524723DOI: 10.1002/age.70170Google Scholar: Lookup

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Summary
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Affiliations
MeSH
References
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Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

This research developed and tested a DNA-based method using a multiplexed SNP-PCR sequencing panel to accurately identify individual Thoroughbred horses from various biological samples, aiding in forensic and doping investigations.

Background and Purpose

Accurately identifying individual horses from biological samples is crucial for:

Resolving ambiguous sample sources.
Providing individual-specific controls for detecting gene editing.
Forensic examinations during potential doping cases.

Traditional methods might be limited by sample types or DNA quality; thus, a reliable, sensitive, and multiplexed method is desirable.

Methodology

The researchers designed a SNP-PCR sequencing panel targeting 93 single nucleotide polymorphism (SNP) markers in a single reaction.
They used 264 Thoroughbred horses’ whole blood samples—collected routinely for doping tests—to evaluate:

The panel’s ability to amplify markers consistently.
The panel’s discriminating power (how well it distinguishes individuals) in a population context.

Sensitivity tests included:

Dilution series to determine the lowest DNA concentration yielding accurate results.
Testing across different sample matrices, such as various types of blood specimens, hair, and feces.

Key Findings

The 93-marker SNP panel showed a high amplification rate and robust discriminating power among the Thoroughbred population.
No genotyping errors due to allelic dropout occurred down to DNA concentrations as low as 0.25 ng/μL.
Blood samples collected with different anticoagulants (EDTA and lithium heparin) performed similarly, indicating flexibility in sample collection methods.
Forensic-type samples, such as blood stains on miscellaneous surfaces (e.g., a blood tube label), produced reliable genotypes, although some lower DNA concentration samples had more variable success.

Practical Demonstration

A blind test mimicking a forensic case was conducted involving 10 “suspect” DNA samples and one unknown test blood sample.
The analyst was able to correctly identify the source of the test sample, showcasing the panel’s effectiveness in real-world scenarios.

Implications and Applications

This SNP-PCR panel provides a reliable and sensitive method for Thoroughbred horse identification, which can be employed in:

Doping control laboratories.
Forensic investigations relating to horse samples.
Validation and monitoring of gene editing.

The adaptability across sample types enhances its utility for cases where sample integrity or availability is compromised.
The method supports the integrity and fairness of Thoroughbred racing and breeding by enabling precise genetic identification and verification.

Cite This Article

APA

Maniego J, Harding C, Hincks P, Habershon-Butcher J, Ryder E. (2026). Approaches to Thoroughbred Horse Identification in Diverse Sample Types Using Multiplexed SNP-PCR Sequencing Panels. Anim Genet, 57(4), e70170. https://doi.org/10.1002/age.70170

Publication

Animal genetics

ISSN: 1365-2052

NlmUniqueID: 8605704

Country: England

Language: English

Volume: 57

Issue: 4

Pages: e70170

Researcher Affiliations

Maniego, Jillian

LGC Ltd, Fordham, Cambridgeshire, UK.

Harding, Caitlin

LGC Ltd, Fordham, Cambridgeshire, UK.

Hincks, Pamela

LGC Ltd, Fordham, Cambridgeshire, UK.

Habershon-Butcher, Jocelyn

British Horseracing Authority, London, UK.

Ryder, Edward

LGC Ltd, Fordham, Cambridgeshire, UK.

MeSH Terms

Animals
Horses / genetics
Polymorphism, Single Nucleotide
Genotype
Sequence Analysis, DNA / veterinary
Multiplex Polymerase Chain Reaction / veterinary
Multiplex Polymerase Chain Reaction / methods
Genotyping Techniques / veterinary
Polymerase Chain Reaction / veterinary

References

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