Microhaplotypes are an emerging type of forensic genetic marker that are expected to support multiple forensic applications. Here, we developed a 124-plex panel for microhaplotype genotyping based on next-generation sequencing (NGS). The panel yielded intralocus and interlocus balanced sequencing data with a high percentage of effective reads. A full genotype was determined with as little as 0.1 ng of input DNA. Parallel mixture experiments and in-depth comparative analyses were performed with capillary-electrophoresis-based short tandem repeat (STR) and NGS-based microhaplotype genotyping, and demonstrated that microhaplotypes are far superior to STRs for mixture deconvolution. DNA from Han Chinese individuals (n = 256) was sequenced with the 124-plex panel. In total, 514 alleles were observed, and the forensic genetic parameters were calculated. A comparison of the forensic parameters for the 20 microhaplotypes with the top A e values in the 124-plex panel and 20 commonly used forensic STRs showed that these microhaplotypes were as effective as STRs in identifying individuals. A linkage disequilibrium analysis showed that 106 of the 124 microhaplotypes were independently hereditary, and the combined match probability for these 106 microhaplotypes was 5.23 × 10 −66. We conclude that this 124-plex microhaplotype panel is a powerful tool for forensic applications. The microhaplotype is a powerful new type of forensic genetic marker 1,2. It is the combination of two or more closely linked single-nucleotide polymorphisms (SNPs) within DNA segments of 200 base pairs (bp), and offers multiple forensic applications 3-7. Short tandem repeat (STR) genotyping is currently the dominant technology in forensic DNA laboratories. Although it works well with single-sourced DNA samples, great challenges are encountered with DNA mixtures because stutters in the major donor DNA can be indistinguishable from alleles in the minor donor DNA 3,8. Stutters are unavoidable during the replication of repetitive DNA, and they severely interfere with mixture deconvolution. SNPs are not repetitive sequences, but are typically biallelic, which restricts their utility in the analysis of mixtures. Microhaplotypes have the advantages of both STRs and SNPs because they are multiallelic and do not produce stutters during amplification. Therefore, microhaplotypes are perfect genetic markers for mixture deconvolution. Although capillary electrophoresis (CE)-based genetic analyzers are widely used in forensic DNA laboratories, these machines are unsuitable for microhaplotype genotyping 8. Several methods have been used for microhaplotype detection. TaqMan assays have been used to type each SNP that constitutes a microhaplotype 8 , followed by a PHASE software analysis to determine the cis/trans relationships between individual SNP alleles. Single-strand conformational polymorphisms 9 and high-resolution melting curves 4 have also been used for microhaplotype genotyping. These methods are simple and inexpensive, but they can pose problems when multipl...

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