The forensic analysis of DNA mixtures remains challenging due to extensive allele sharing among contributors, which severely limits the ability to distinguish individual profiles, particularly in high-ratio or degraded samples. A key strategy to address this is increasing the information content per genetic marker, thereby enhancing polymorphism and reducing ambiguity in contributor assignment. Microhaplotype markers, which combine multiple tightly linked SNPs within short amplicons (<300 bp), represent a significant advancement in this field.

With the development of long-read sequencing, the microhaplotype concept can now be extended beyond traditional length constraints. By spanning longer genomic regions and incorporating a greater number of linked variants within a single locus, the overall allelic diversity can be substantially increased. Based on this concept, we propose extended microhaplotypes, a novel class of high-informativeness markers. 

To assess their forensic utility, we developed an ONT-based process. Candidate loci were identified from the 1000 Genomes Project (Southern Han Chinese population) and refined using stringent bioinformatic filters, including requirements for amplicon length (300–800 bp), minimum SNP density (≥3 SNPs per locus), high minor allele frequency, and absence of homologous regions. A final panel of 146 autosomal extended microhaplotypes was validated in 125 individuals. Ae values ranged from 1.187 to 14.003, with an average of 4.61. Notably, 84.2% of loci Ae > 3, 33.6% Ae > 5, and 9.5% Ae > 7.

Compared to conventional forensic markers, extended microhaplotypes enhance the accuracy of contributor number (NOC) inference in DNA mixtures by providing a greater number of distinguishable, haplotype-resolved alleles per locus and mitigating ambiguity caused by allele sharing. The phased architecture supports robust likelihood ratio (LR)-based weight-of-evidence assessment under mixture deconvolution models, underscoring their utility in complex forensic casework.

In summary, extended microhaplotypes offer a scalable solution to complex forensic scenarios, providing a robust framework for DNA analysis and effectively addressing the critical limitations of current marker systems in complex mixtures.

Authors

• Peng Bai (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Peiyu Wang (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Mengyu Tan (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Shengqiu Qu (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)
• Weibo Liang (Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, China)