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

Area of Science:

• Veterinary Genetics
• Forensic Science
• Molecular Biology

Background:

• Cattle paternity testing currently relies on dinucleotide STR (di-STR) or SNP analysis.
• Di-STR markers often yield stutter artifacts, impacting genotyping accuracy.
• Single nucleotide polymorphisms (SNPs) lack sufficient polymorphic levels for reliable identification.

Purpose of the Study:

• To develop and validate a novel integrated next-generation sequencing (NGS) panel for cattle.
• To overcome the limitations of existing cattle DNA testing methods.
• To enhance the accuracy and efficiency of cattle genetic profiling for forensic applications.

Main Methods:

• Developed an NGS panel integrating 33 di-STRs and 30 tetranucleotide STRs (tetra-STRs).
• Included mitochondrial displacement loop (D-loop) region, cytochrome b (CYTB), and recombination-activating gene 1 (RAG1) in the panel.
• Validated the panel using blood and hair samples, assessing genotyping success, amplification efficiency, and consistency.

Main Results:

• Achieved a high average genotyping success rate of 99.6%.
• Demonstrated balanced amplification efficiency across most STR loci.
• Reported an average genotyping consistency rate of 96.4% between blood and hair samples.
• Attained high discrimination power (1-1.8 × 10^-13) and cumulative probability of exclusion (1-1.7 × 10^-8) for duos.

Conclusions:

• The novel NGS panel offers a significant advancement over current capillary electrophoresis (CE)-based systems for cattle profiling.
• The panel provides superior accuracy, multiplex capability, and efficiency for forensic genetic testing in cattle.
• Demonstrated applicability and reliability in resolving cattle paternity disputes through casework analysis.