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.