Developmental and validation of a novel small and high-efficient panel of microhaplotypes for forensic genetics by the next generation sequencing

Abstract

Background: In the domain of forensic science, the application of kinship identification and mixture deconvolution techniques are of critical importance, providing robust scientific evidence for the resolution of complex cases. Microhaplotypes, as the emerging class of genetic markers, have been widely studied in forensics due to their high polymorphisms and excellent stability.

Results and discussion: In this research, a novel and high-efficient panel integrating 33 microhaplotype loci along with a sex-determining locus was developed by the next generation sequencing technology. In addition, we also assessed its forensic utility and delved into its capacity for kinship analysis and mixture deconvolution. The average effective number of alleles (Ae) of the 33 microhaplotype loci in the Guizhou Han population was 6.06, and the Ae values of 30 loci were greater than 5. The cumulative power of discrimination and cumulative power of exclusion values of the novel panel in the Guizhou Han population were 1-5.6 × 10^{- 43} and 1-1.6 × 10^{- 15}, respectively. In the simulated kinship analysis, the panel could effectively distinguish between parent-child, full-sibling, half-sibling, grandfather-grandson, aunt-nephew and unrelated individuals, but uncertainty rates clearly increased when distinguishing between first cousins and unrelated individuals. For the mixtures, the novel panel had demonstrated excellent performance in estimating the number of contributors of mixtures with 1 to 5 contributors in combination with the machine learning methods.

Conclusions: In summary, we have developed a small and high-efficient panel for forensic genetics, which could provide novel insights into forensic complex kinships testing and mixture deconvolution.

Keywords: Complex kinships; Forensic genetics; Guizhou Han; Microhaplotype; Next generation sequencing.

Fig. 1

General information of selected 33 microhaplotype loci. Physical positions of selected 33 microhaplotypes in different chromosomes (a) and their forensic parameters in East Asian population (b). The heatmap in the circle diagram was the Ae values of selected 33 microhaplotypes in different continental populations

Fig. 2

Population genetic analyses of 26 reference populations from different continents based on selected 33 microhaplotypes. a, MDS analysis of 26 populations; b, the phylogenetic tree of 26 populations; c, population genetics structure analyses of 26 reference populations

Fig. 3

Sequencing results (depth of coverage and allele coverage ratio) of the 33 microhaplotypes in the Guizhou Han population

Fig. 4

Loci detection rates of 33 microhaplotypes in different mixed ratios of 9948 and 9947 A samples. The hollow circle indicated the missing locus

Fig. 5

Allele frequency distributions (a) and forensic parameters (b) of the 33 microhaplotypes in the Guizhou Han population

Fig. 6

The density plot of Log10(LR) for different relationships. Log10(LR) were drawn for true relationships (blue curve) and true unrelated pairs (pink curves)

Fig. 7

Confusion matrices of predicted and actual results for the number of contributors in testing samples by the Naive Bayes (a), random forest (b), decision tree (c), XGBoost (d), classification and regression trees (e) and linear discriminant analysis methods (f)