Nanopore sequencing of MiniHap biomarkers for forensic DNA mixture deconvolution: A proof-of-principle study

Abstract

Mixture deconvolution remains one of the major challenges in the field of forensic science. Currently, genetic markers are used and studied in the field of forensic genetics, including short tandem repeat (STR), insertion/deletion polymorphism (InDel), single nucleotide polymorphism (SNP), InDel closely linked to STR (DIP-STR), SNP closely linked to STR (SNP-STR), InDel closely linked to SNP (DIP-SNP) and microhaplotype (MH), all of which have been studied for DNA mixture analysis and have their own advantages and disadvantages. Mini-haplotype (MiniHap), as a novel haplotype genetic marker, contains 5 or more SNPs. A previous study has substantiated its significant high polymorphic characteristics, and it is expected to have potential applications in individual identification, paternity testing, ancestry inference, and mixture deconvolution. In this study, we first screened 22 MiniHaps with high polymorphism potential and constructed a panel based on the QNome nanopore sequencing device. Subsequently, we tested 100 unrelated Chinese Han individuals to evaluate the sequencing performance, allele (haplotype) frequencies, effective number of alleles (A_e) and forensic parameters of the 22 MiniHaps markers included in this novel assay. Next, a series of mixture simulations (two- or three-person mixtures with mixing ratios of 1:1–1:99 or 1:1:1–1:8:1) based on three standard materials (9947 A, 9948 and 2800 M) were detected by this MiniHap panel to explore its potential for DNA mixture deconvolution. The average A_e value was 10.9574, and 52.38 % of MiniHap loci had A_e values greater than 12.0000. The mean values of genetic diversity (GD) and power of discrimination (PD) were 0.8717 and 0.9457, respectively. Notably, most MiniHaps (85.71 %) had PD values exceeding 0.9000. The combined match probability (CMP) and combined power of exclusion (CPE) of this MiniHap panel were 4.4505 × 10⁻³¹ and 0.999999999999999996653, respectively. Moreover, the results of mixture analysis demonstrated that this MiniHap panel allowed detecting the components of minor contributor (s) even in imbalanced mixture samples, with detection limits of 1:39 and 1:8:1 for two- and three-person mixtures, respectively. In summary, MiniHap markers have remarkable application potential in mixture deconvolution, and it is necessary to conduct in-depth research on MiniHap markers for mixture analysis in the future.