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...

In the present study, 24 Y-chromosomal short tandem repeat (Y-STR) loci were analyzed in 250 unrelated Hani male individuals from Lvchun county, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, Southwest China. The gene diversity of the 24 Y-STR loci in the studied Hani group ranged from 0.2683 (DYS437) to 0.8837 (DYS447). According to haplotypic analysis of the 24 Y-STR loci, 204 different haplotypes were obtained, 174 of which were unique. The haplotype diversity and discrimination capacity in Hani group were 0.9977 and 0.8160 at 24 STR loci, respectively. Six single non-fraction off-ladder alleles were observed at DYS447 in 103 samples, in addition to the alleles 19 to 28 included in the allelic ladder, alleles 13, 14, 15, 16, 17, and 18 were also observed at DYS447. One intermediate allele 20.2 was observed in one individual at DYS527a/b. We analyzed interpopulation differentiations by making comparisons between Yunnan Hani group and other 17 groups. The results of pairwise genetic distances, multidimensional scaling plot, and neighbor-joining tree at the same set of 17 Y-filer loci indicated that Yunnan Hani group had the closer genetic relationships with Yunnan Han group. The present results may provide useful information for paternal lineages in forensic cases and can also increase our understanding of the genetic relationships between Hani and other groups.

The genetic polymorphisms of 20 autosomal short tandem repeat (STR) loci included in the PowerPlex® 21 kit were evaluated in 522 healthy unrelated Vietnamese from Yunnan, China. All of the loci reached the Hardy-Weinberg equilibrium. These loci were examined to determine allele frequencies and forensic statistical parameters. The combined discrimination power and probability of excluding paternity of the 20 STR loci were 0.999999999999999999999991 26 and 0.999999975, respectively. Results suggested that the 20 STR loci are highly polymorphic, which is suitable for forensic personal identification and paternity testing.

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The brown type donkeys of Andhra Pradesh, which are mainly concentrated in Kurnool and Anathapur districts, were evaluated for within breed genetic diversity and bottlenecks using heterologous microsatellite markers. The genomic DNA, isolated from 28 blood samples collected from Kurnool district, were amplified by PCR using FAM and HEX labeled primers and resolved for alleles on automatic DNA sequencer. In all, twenty loci of the horse origin were tested and only 12 loci gave scorable results. Rest of the loci either did not amplify (HMS3 and HMS7) or did not resolve properly (VHL20) or showed less than 4 alleles (HMS5, HMS6, HTG4, ASB17 and COR22) in the studied population. At the 12 loci included in the final analysis, the PCR product size range varied from 76-92 bp at locus HTG6 to 257-273 bp at locus COR18. The observed number of alleles varied from 4 (VHL209) to 10 (AHT5 and HTG7) with a mean of 6.92±1.83. The effective number of alleles ranged from 1.62 (VHL209) to 7.91 (AHT5) with a mean of 4.21±2.06. The observed heterozygosity ranged from 0.32 (HMS2) to 0.92 (AHT5) with a mean of 0.57±0.2. The expected heterozygosity ranged between 0.39 (VHL209) to 0.89 (AHT5 and HTG7) with a mean of 0.72±0.14. The mean genetic diversity estimate (FIS) was 0.21 indicating moderately high levels of inbreeding. The cumulative exclusion probability (PE) of these loci was 0.999892 indicating their suitability for parentage testing in these donkeys. The sign test, standardized differences test, the Wilcoxon test using the allelic frequency data at the studied loci as well as normal 'L' shaped distribution of the allelic frequency indicated the absence of any recent genetic bottleneck in Brown type donkeys of Andhra Pradesh. When these donkeys were compared to Spiti donkeys of Himachal Pradesh on the basis of allelic frequency data at these loci, they showed Nei's standard and unbiased genetic distances of 0.32 and 0.29, respectively.