MHappaMundi: A custom AmpliSeq microhaplotype panel for ancestry inference

Recent advancements in massively parallel sequencing technologies have led to the exploration of a new class of genetic markers called microhaplotypes (MHs). MHs exhibit unique characteristics that highlight their potential as viable alternatives to STRs and SNPs in addressing challenges commonly faced in forensic investigations. Different studies have detailed the development of MH panels for distinct purposes in forensics (e.g., human identification, mixture deconvolution, kinship testing, or biogeographic ancestry inference). However, MH panels with the sole purpose of ancestry inference are scarce and show some limitations that hinder a broader acceptance within the community. A new ancestry-informative MH panel, named MHappaMundi, was developed with the specific aim of differentiating Sub-Saharan African, European, South Asian, East Asian, and Native American ancestries. The selection method involved computing pairwise F_ST for all the MHs present in the MicroHapDB database between all pairs of continental groups, based on the 1000 Genomes Project (1kGP) populations. This approach aimed to establish a final panel ensuring balanced genetic distances between each population pair. From this selection method, 100 MHs were recruited for the panel. The performance of MHappaMundi was assessed by sequencing 147 European (Danes and Portuguese), 120 East Asian (Mainland and Okinawan Japanese), and 101 Native American (Brazilian and Ecuadorian) individuals on the Ion GeneStudio™ S5 System. A total of 8 loci were excluded due to insufficient read depth or technical inconsistencies. The final panel included 92 MHs and 466 allele-defining SNPs. Population genetic clustering was evaluated through STRUCTURE analysis, Principal Component Analysis, and Multidimensional scaling for the genotyped populations from this study and available online data from the 1kGP and Middle Eastern populations. In these analyses, the panel demonstrated great effectiveness in separating the five continental groups, particularly the Europeans and South Asians, which had presented challenges in previous MH ancestry panels. Moreover, the panel revealed an effective estimation of ancestry proportions in admixed individuals. In conclusion, the MHappaMundi serves as a potential asset for ancestry inference in forensic and population genetics.