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

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.