Integrated Microfluidic System for Rapid 89-Plex Y-SNP Profiling: Development and Forensic Validation

Area of Science:

• Forensic Genetics
• Microfluidics
• Molecular Biology

Background:

• Current Y-chromosomal single-nucleotide polymorphism (Y-SNP) detection methods are often cumbersome and not field-adaptable.
• There is a need for portable, automated solutions for Y-SNP analysis in forensic applications.

Purpose of the Study:

• To develop and validate an 89-plex microfluidic Y-SNP system for rapid, on-site genotyping.
• To create a fully automated workflow from sample to result, enhancing forensic analysis capabilities.

Main Methods:

• Development of a microfluidic system with disposable DNA extraction/amplification and capillary electrophoresis chips.
• Integration of in situ lyophilized reagents for long-term storage and a portable device for automation.
• Design of two panels (AIYSNP42 and AIYSNP47) based on the ISOGG phylogenetic tree for multi-level haplogroup resolution.

Main Results:

• Achieved a fully automated "sample-in-answer-out" workflow, completing 89-locus Y-SNP genotyping in 139 minutes.
• Demonstrated high sensitivity (2.5 ng DNA), success rate (93.6% across 94 samples), and robustness against inhibitors and DNA degradation.
• Showcased excellent reproducibility (CV < 0.5%) and reliable detection of male DNA in mixtures (≥2% in male-female, ≥33% in male-male).

Conclusions:

• The developed microfluidic system significantly reduces reliance on traditional laboratory workflows.
• Enables rapid, on-site Y-SNP analysis for diverse forensic applications including pedigree tracing, ancestry inference, and mixture interpretation.