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• A microfluidic-based SNP genotyping (MSG) chip was established to detect 72 SNPs for ancestry inference. • The detection process took less time (2.5 h) and was simpler than other SNP genotyping methods. • The performance of the chipincluing accuracy, specificity, and sensitivity was fully evaluated. • The MSG chip can be used in ancestry inference in forensic practice. In forensics, ancestry inference can provide leads for criminal investigation. Therefore, the time needed to generate results is the first priority. Single nucleotide polymorphisms (SNPs) are widely used genetic markers for ancestry inference. In this study, a rapid microfluidic-based SNP genotyping (MSG) chip was established to detect 72 autosomal SNPs for ancestry inference of East Asian populations. The DNA template was infused into the chip and distributed into reaction chambers with pre-spotted primer pairs under centrifugation. After sealing the inlets/outlets and the connections among adjacent reaction chambers, the chip was placed onto a plate thermocycler for competitive allele-specific PCR. The SNP genotyping results were generated by analyzing the extracted fluorescence signal of each reaction chamber after PCR was completed. The detection process took less time (2.5 h) and was simpler than other SNP genotyping methods, such as SNaPshot and MassArray. To assess the performance of the chip, its accuracy, specificity, and sensitivity were evaluated. A total of 50 samples were genotyped using the chip, and the consistency of all of the typing and sequencing results was 100%. For ancestry inference of unknown individuals, a reference database of 3628 individuals from 57 populations was employed, and the ancestry origin of 110 test samples was inferred, demonstrating that the differentiation of East Asian subpopulations can be achieved through the MSG chip method. Overall, the MSG chip method established in this study can effectively be used for rapid and accurate ancestry inference. [ABSTRACT FROM AUTHOR]

Inferring an unknown DNA's ancestry using a set of ancestry-informative single nucleotide polymorphisms (SNPs) in forensic science is useful to provide investigative leads. This is especially true when there is no DNA database match or specified suspect. Thus, a set of SNPs with highly robust and balanced differential power is strongly demanded in forensic science. In addition, it is also necessary to build a genotyping database for estimating the ancestry of an individual or an unknown DNA. For the differentiation of Africans, Europeans, East Asians, Native Americans, and Oceanians, the Global Nano set that includes just 31 SNPs was developed by de la Puente et al. Its ability for differentiation and balance was evaluated using the genotype data of the 1000 Genomes Phase III project and the Stanford University HGDP-CEPH. Just 402 samples were genotyped and analyzed as a reference set based on statistical methods. To validate the differentiating capacity using more samples, we developed a single-tube 28-plex SNP assay in which the SNPs were chosen from the 31 allelic loci of the Global AIMs Nano set. Three tri-allelic SNPs used to differentiate mixed-source DNA contribute little to population differentiation and were excluded here. Then, 998 individuals from 21 populations were typed, and these genotypes were combined with the genotype data obtained from 1000 Genomes Phase III and the Stanford University HGDP-CEPH (3090 total samples,43 populations) to estimate the power of this multiplex assay and build a database for the further inference of an individual or an unknown DNA sample in forensic practice. [ABSTRACT FROM AUTHOR]