Integrated Genetic Profiling for Cattle Parentage Testing via Next‐Generation Sequencing

Cattle are frequently involved in civil disputes and offenses. Currently, the paternity testing of cattle is mainly done by analysis of dinucleotide STR (di‐STR) or SNPs. However, the di‐STR marker usually produces many stutter artifacts that affect the accurate genotyping, and the polymorphic level of SNPs is not high enough. To overcome these limitations, we developed and validated a novel integrated next‐generation sequencing (NGS) panel for cattle. It comprises 33 reported di‐STRs and 30 newly identified tetranucleotide STRs (tetra‐STRs), the mitochondrial displacement loop (D‐loop) region, as well as cytochrome b (CYTB) and recombination‐activating gene 1 (RAG1). The results showed a high genotyping success rate (99.6% on average) and balanced amplification efficiency across most STR loci. In addition, the panel's average genotyping consistency rate reached 96.4% between blood and hair samples. The total discrimination power and cumulative probability of exclusion for duos of this panel were 1–1.8 × 10−13 and 1–1.7 × 10−8, respectively. Application in paternity testing caseworks demonstrates its applicability and reliability for resolving paternity disputes. Overall, this study provided a cattle profiling NGS panel with a substantial advancement over current capillary electrophoresis (CE)‐based systems, offering superior accuracy, multiplex capability, and efficiency for forensic genetic testing in cattle.