Short tandem repeats (STRs) are the most widely used genetic markers in forensic application, but they are not ideal genetic markers for the analysis of forensic challenging samples such as highly degraded or unbalanced mixed samples because of their relatively large amplicons and stutter peaks. In this study, we developed a set of short microhaplotypes based on non-binary SNPs with molecular extent sizes no longer than 60 bases and genotyped 100 unrelated individuals from northern Han groups. Our results showed this panel has similar discrimination power to STR kits, as the combined random match probability (CMP) reached 1.396 × 10 and mean effective number of alleles (Ae) was 3.59. The cumulative probability of exclusion for duos (CPE-duos) was 0.999919 and the cumulative probability of exclusion for trios (CPE-trios) was 0.9999999987, suggesting this panel could be applied for forensic personal identification and parentage testing independently. Population differentiation in 26 populations from the 1000 Genomes Project indicated this panel could distinguish populations from Africa, East Asia, South Asia, America, and Europe. These microhaplotypes based on non-binary SNPs have short amplicons, good discrimination power, no stutter artifacts, and have great potential in detection of highly degraded and unbalanced mixtures for personal identification, paternity testing, and ancestry inference.
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