Establishment and Application of a 42-plex Microhaplotype Assay in Forensic Genetics

Area of Science:

• Forensic Genetics
• Molecular Biology
• Genetics

Background:

• Microhaplotypes (MHs) are increasingly utilized in forensic genetics due to their high discriminatory power.
• Next-generation sequencing (NGS) platforms offer enhanced capabilities for analyzing complex genetic markers.
• Establishing robust multiplex assay systems is crucial for efficient forensic casework.

Purpose of the Study:

• To develop and forensically validate a 42 microhaplotypes (MHs) multiplex assay system using NGS.
• To assess the practical application of this MHs assay in forensic genetics.
• To evaluate the system's performance characteristics, including repeatability, sensitivity, specificity, stability, and mixture analysis capabilities.

Main Methods:

• Selection of 42 highly polymorphic microhaplotypes (MHs).
• Sequencing using the MiSeq FGx™ platform to validate assay performance.
• Population genetic analysis of 102 unrelated Chinese Han individuals.

Main Results:

• The 42-plex MHs assay demonstrated 100% repeatability and high sensitivity (down to 0.0625 ng).
• The system exhibited robustness against common forensic inhibitors (indigo, humic acid, hemoglobin, urea).
• Effective detection of mixtures involving up to four individuals was achieved, with high combined powers of discrimination (1-3.45×10⁻³⁰) and exclusion (1-3.77×10⁻¹¹).

Conclusions:

• The established 42-plex MHs assay is highly repeatable, sensitive, and stable, with excellent mixture analysis capabilities.
• This MHs system possesses significant polymorphism, making it valuable for forensic individual identification and paternity testing.
• The validated assay is well-suited for practical applications in forensic genetics.