Screening and selection of 21 novel microhaplotype markers for ancestry inference in ten Chinese subpopulations

Abstract

Genetic findings suggested ethnolinguistically diverse populations in China harbor differentiated genetic structure and complex evolutionary admixture histories, providing the genetic basis and theoretical foundation for forensic biogeographical ancestry inference (BGAI). Forensic assays for BGAI among intracontinental eastern Eurasians were previously conducted mainly based on SNPs or InDels. Microhaplotypes, as a set of closely linked SNPs within 200 base pairs, possess the advantages of both STRs and SNPs and have great potential in forensic ancestry inference. However, the forensic assay developed based on ancestry-informative microhaplotypes in the BGAI remains to be further explored, especially in China, which has rich genetic diversity. We described a new BGAI panel based on 21 novel identified ancestry-informative microhaplotypes that focused on dissected finer-scale ancestry compositions of Chinese populations. We initially screened all possible microhaplotypes with high Fst values among five East Asian populations and finally employed 21 candidate microhaplotypes in two multiplex SNaPshot assays. Forensic amplification efficiency and statistically/physically phased haplotypes of the 21 microhaplotypes were validated using SNaPshot and massively parallel sequencing (MPS) platforms. Next, we validated the efficiency of these microhaplotypes for BGAI in 764 individuals from ten Chinese populations using SNaPshot technology. The fine-scale ancestry source and ancestry proportion estimated by principal component analysis (PCA), multidimensional scaling (MDS), phylogenetic tree and model-based STRUCTURE among worldwide populations and East Asians showed that our customized panel could provide a higher discrimination resolution in both continental population stratification and East Asian regional substructure. East Asian populations could be classified into linguistically/geographically different intracontinental subpopulations (Tibeto-Burman, Tai-Kadai and others). Finally, we obtained a higher estimated accuracy using training and tested datasets in the microhaplotype-based panel than traditional SNP-based panels. Generally, the above results demonstrated that this microhaplotype panel was robust and suitable for forensic BGAI in Chinese populations, which provided high discriminatory power for continental populations and discriminated East Asians into linguistically restricted subpopulations.