Mixture deconvolution by massively parallel sequencing of microhaplotypes.

Area of Science:

• Forensic Science
• Genetics
• Molecular Biology

Background:

• Short tandem repeat polymorphisms (STRs) are standard for human identification but have limitations with complex mixtures, including stutter fragments and allele drop-out.
• Massively parallel sequencing (MPS) offers higher discrimination and access to new markers like microhaplotypes (MHs).
• Microhaplotypes are short, highly polymorphic loci with alleles of identical length, producing no stutter fragments, making them promising for mixture analysis.

Purpose of the Study:

• To practically demonstrate the utility of microhaplotypes for forensic mixture interpretation.
• To evaluate a panel of 36 microhaplotypes using MPS for human identification.
• To compare the performance of MPS with microhaplotypes against traditional STR analysis in complex mixtures.

Main Methods:

• A panel of 36 microhaplotypes was tested on the Ion S5™ MPS platform.
• Samples included single-source DNA, synthetic mixtures (2-6 contributors), and casework-like samples.
• The microhaplotype panel was analyzed both multiplexed with STRs/SNPs and individually.

Main Results:

• Single-source sample analysis showed good allele coverage ratios, comparable to STRs analyzed by capillary electrophoresis (CE).
• Mixture studies demonstrated higher input DNA tolerance and increased sensitivity for minor contributors in imbalanced mixtures due to the absence of stutter fragments.
• MPS of microhaplotypes detected more minor donor alleles in casework-like samples compared to MPS and CE of STRs.

Conclusions:

• Massively parallel sequencing of microhaplotypes effectively complements STR analysis in forensic human identification.
• Microhaplotypes significantly enhance the interpretation of complex and imbalanced DNA mixtures.
• This approach offers improved sensitivity and reliability for forensic casework.