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.