Facilitating complex DNA mixture interpretation by sequencing highly polymorphic haplotypes.

Area of Science:

• Forensic genetics
• Genomic analysis
• DNA mixture interpretation

Background:

• Interpreting complex DNA mixtures (≥4 contributors) is challenging for forensic genetics.
• Standard short tandem repeat (STR) markers struggle with higher-order mixtures due to shared alleles.
• Emerging markers like SNPs and short haplotypes lack sufficient polymorphism for complex mixtures.

Purpose of the Study:

• To develop and validate a new panel of highly polymorphic haplotype markers for complex DNA mixture analysis.
• To address the limitations of current forensic DNA typing methods in higher-order mixtures.
• To assess the robustness of a novel allele calling method under high sequencing error rates.

Main Methods:

• Utilized a novel panel of highly polymorphic haplotype markers, suitable for standard or nanopore sequencing (MinION).
• Developed and simulated a method for haplotype allele calling robust to high sequencing error rates.
• Calculated likelihood ratios (LR) to assess individual contribution to complex mixtures.

Main Results:

• The developed method accurately identifies correct alleles in complex mixtures despite high sequencing error rates.
• Contributors to mixtures (up to five individuals) received high likelihood ratios (>10^9).
• Non-contributors were effectively excluded with very low likelihood ratios (<1 in most cases).

Conclusions:

• The proposed haplotype marker system and allele calling method offer a robust solution for complex DNA mixture interpretation in forensics.
• This approach enhances the statistical power for including contributors and excluding non-contributors in challenging forensic samples.
• The method shows promise for improving the accuracy and reliability of forensic DNA analysis, particularly with advanced sequencing technologies.