Internal validation of the GA118-24B Genetic Analyzer, a stable capillary electrophoresis system for forensic DNA identification.

The GA118-24B Genetic Analyzer (hereafter, "GA118-24B") is an independently developed capillary electrophoresis instrument. In the present research, we designed a series of validation experiments to test its performance at detecting DNA fragments compared to the Applied Biosystems 3500 Genetic Analyzer (hereafter, "3500"). Three commercially available autosomal short tandem repeat multiplex kits were used in this validation. The results showed that GA118-24B had acceptable spectral calibration for three kits. The results of accuracy and concordance studies were also satisfactory. GA118-24B showed excellent precision, with a standard deviation of less than 0.1 bp. Sensitivity and mixture studies indicated that GA118-24B could detect low-template DNA and complex mixtures as well as the results generated by 3500 in parallel experiments. Based on the experimental results, we set specific analytical and stochastic thresholds. Besides, GA118-24B showed superiority than 3500 within certain size ranges in the resolution study. Instead of conventional commercial multiplex kits, GA118-24B performed stably on a self-developed eight-dye multiplex system, which were not performed on 3500 Genetic Analyzer. We compared our validation results with those of previous research and found our results to be convincing. Overall, we conclude that GA118-24B is a stable and reliable genetic analyzer for forensic DNA identification.

A preliminary exploration for co-detecting RNA virus and STR type on capillary electrophoresis in forensic practice.

RNA virus infection such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection shows severe respiratory symptoms on human and could be an obvious individual characteristic for investigations in forensic science. As for biological samples suspected to contain RNA virus in forensic casework, it requires respective detection of viral RNA and human DNA: reverse transcriptase polymerase chain reaction and DNA type (short tandem repeat [STR] analysis). Capillary electrophoresis (CE) has been shown to be a versatile technique and used for a variety of applications, so we preliminarily explored the co-detection of RNA virus and STR type on CE by developing a system of co-detecting SARS-CoV-2 and STR type under ensuring both the efficiency of forensic DNA analysis and safety of the laboratory. This study investigated the development and validation of the system, including N and ORF1ab primer designs, polymerase chain reaction amplification, allelic ladder, CE detection, thermal cycling parameters, concordance, sensitivity, species specificity, precision, and contrived and real SARS-CoV-2 sample studies. Final results showed the system could simultaneously detect SARS-CoV-2 and STR type, further indicating that CE has possibilities in the multi-detection of RNA viruses/STR type to help to prompt individual characteristics (viral infection) and narrow the scope of investigation in forensic science.

Preliminary study on genetic factors related to Demirjian's tooth age estimation method based on genome-wide association analysis.

The age determination of individuals, especially minors, is critical in forensic research. In forensic practice, dental age estimation is one of the most commonly used methods for determining age as teeth are easy to preserve and relatively resistant to environmental factors. Tooth development is affected and regulated by genetic factors; however, these are not incorporated into current commonly used tooth age inference methods, leading to unreliable results. Here, we established a Demirjian and a Cameriere tooth age estimation-based methods suitable for use in children in southern China. By using the difference between the inferred age and the actual age (MD) as the phenotype, we identified 65 and 49 SNPs related to tooth age estimation from 743,722 loci among 171 children in southern China through a genome-wide association analysis (p<0.0001). We also conducted a genome-wide association study on dental development stage (DD) using the Demirjian tooth age estimation method and screened two sets of SNP sites (52 and 26) based on whether age difference was considered. The gene function enrichment analysis of these SNPs found that they were related to bone development and mineralization. Although SNP sites screened based on MD seem to improve the accuracy of tooth age estimation, there is little correlation between these SNPs and an individual's Demirjian morphological stage. In conclusion, we found that individual genotypes can affect tooth age estimation, and based on different phenotypic analysis models, we have identified some novel SNP sites related to tooth age inference and Demirjian's tooth development stage. These studies provide a reference for subsequent phenotypic selection based on tooth age inference analysis, and the results could possibly be used in the future to make forensic age estimation more accurate.

Ventilation in a Residential Building Brings Outdoor NOx Indoors with Limited Implications for VOC Oxidation from NO3 Radicals.

Energy-efficient residential building standards require the use of mechanical ventilation systems that replace indoor air with outdoor air. Transient outdoor pollution events can be transported indoors via the mechanical ventilation system and other outdoor air entry pathways and impact indoor air chemistry. In the spring of 2022, we observed elevated levels of NOx (NO + NO2) that originated outdoors, entering the National Institute of Standards and Technology (NIST) Net-Zero Energy Residential Test Facility through the mechanical ventilation system. Using measurements of NOx, ozone (O3), and volatile organic compounds (VOCs), we modeled the effect of the outdoor-to-indoor ventilation of NOx pollution on the production of nitrate radical (NO3), a potentially important indoor oxidant. We evaluated how VOC oxidation chemistry was affected by NO3 during NOx pollution events compared to background conditions. We found that nitric oxide (NO) pollution introduced indoors titrated O3 and inhibited the modeled production of NO3. NO ventilated indoors also likely ceased most gas-phase VOC oxidation chemistry during plume events. Only through the artificial introduction of O3 to the ventilation duct during a NOx pollution event (i.e., when O3 and NO2 concentrations were high relative to typical conditions) were we able to measure NO3-initiated VOC oxidation products, indicating that NO3 was impacting VOC oxidation chemistry.

Application of Microhaplotypes in Sibling Kinship Testing.

OBJECTIVES: To investigate the efficacy of different numbers of microhaplotype (MH) loci and the introduction of different reference samples on the identification of full sibling, half sibling and differentiation between full sibling and half sibling kinships, and to explore the effect of changing mutation rate on sibling testing. METHODS: First, a family map involving three generations was established, and four full sibling identification models, five half sibling identification models and five models distinguishing full and half siblings were constructed for different reference samples introduced. Based on the results of the previous study, two sets of nonbinary SNP-MH containing 34 and 54 loci were selected. Based on the above MH loci, 100 000 pairs of full sibling vs. unrelated individuals, 100 000 pairs of half sibling vs. unrelated individuals and 100 000 pairs of full sibling vs. half sibling were simulated based on the corresponding sibling kinship testing models, and the efficacy of each sibling kinship testing model was analyzed by the likelihood ratio algorithm under different thresholds. The mutant rate of 54 MH loci was changed to analyze the effect of mutation rate on sibling identification. RESULTS: In the same relationship testing model, the systematic efficacy of sibling testing was positively correlated with the number of MH loci detected. With the same number of MH loci, the efficacy of full sibling testing was better than that of uncle or grandfather when the reference sample introduced was a full sibling of A, but there was no significant difference in the identification efficacy of the four reference samples introduced for full sibling and half sibling differentiation testing. In addition, the mutation rate had a slight effect on the efficacy of sibling kinship testing. CONCLUSIONS: Increasing the number of MH loci and introducing reference samples of known relatives can increase the efficacy of full sibling testing, half sibling testing, and differentiation between full and half sibling kinships. The level of mutation rate in sibling testing by likelihood ratio method has a slight but insignificant effect on the efficacy.

Assess the diversity of gut microbiota among healthy adults for forensic application.

BACKGROUND: Human gut microbiota is individually unique that hints the microbiota in fecal traces left in the crime scene could act as a potential biomarker for forensic personal identification. Next-generation DNA sequencing and bioinformatic analysis of fecal samples are revolutionizing our insights into gut microbial communities. While the formation of the gut microbiota is known to be multifactorial, it is unclear whether these characteristics can be applied to forensic applications. Therefore, the gut microbiota of healthy adults with different traits was investigated in this study. RESULTS: Based on the STAMP analysis of each study group, the difference in gut microbiota composition of male and female subjects was observed. The male group was characterized by taxa in the phylum Proteobacteria, while the female group was described by Synergistetes phylum. The gut bacterial community assembly mechanism was mainly affected by the deterministic process. In addition, gut microbiota composition showed meaningful discrimination in each of the BMI groups. At the phylum level, in male subjects, increased representative phyla were Patescibacteria (p < 0.05) in the underweight group and Bacteroidetes (p < 0.05) in the normal-weight group, while in the female group, the significantly different phyla were Bacteroidetes, Firmicutes, and Actinobacteria. At the genus level, 44 unique genera were found to be significantly distinct across BMI study groups. By Fisher's Linear Discriminant Analysis, ninety-four point four percent (94.4%) of original BMI grouped subjects were correctly classified. The linear regression analysis model showed an accuracy of seventy-four percent (74%) in predicting body type. CONCLUSION: In conclusion, subjects with different individual characters have specific gut microbiota, and can be discriminated by bioinformatics methods, suggesting it is promising to apply gut microbiota to forensic personal identification.

Genetic structure and forensic characteristics of the Kyrgyz population from Kizilsu Kirghiz autonomous prefecture based on autosomal DIPs.

Living in the heart of Eurasia, the Kyrgyz ethnic minority have a complex human evolutionary and migration history. However, the genetic architecture of the Kyrgyz population has not been fully explored. We studied 526 Kyrgyz samples from Kizilsu Kirghiz Autonomous Prefecture in Xinjiang using the Investigator® DIPplex kit. All loci followed Hardy-Weinberg equilibrium (HWE). The combined power of discrimination (CPD) and combined power of paternity exclusion (CPE) was 0.9999999999988 and 0.9936, respectively. Compared with 90 reference populations, five InDels (HLD99, HLD81, HLD64, HLD118, and HLD111) have the potential to distinguish the Kyrgyz/Uyghur/Kazak population from other East Asian populations. Our results suggested a close genetic relationship between the Kyrgyz population and the Uyghur/Kazak populations, followed by South Asian populations. This was in accordance with the inland migration hypothesis or modern human migration influenced by warfare. Overall, this system can be used as a powerful tool in forensic individual identification and as a complementary tool in paternity cases and biogeographic ancestry analyses.

Development and validation of a novel 26-plex system for prenatal diagnosis with forensic markers.

Short tandem repeat (STR) loci are commonly used in forensic casework, such as personal identification and paternity testing. In recent years, STR has also been widely used for rapid, accurate and automated prenatal diagnosis, known as quantitative fluorescent PCR (QF-PCR). Despite their usefulness, the current systems often lack the power to detect mosaicism for Turner syndrome. In this study, we developed a novel 26-plex system that combined the 22 STRs in chromosome 21/18/13/X, 3 sex loci and 1 quality control marker (TAF9L). The system was generated to achieve greater diagnostic power of trisomy 21/18/13 and sex chromosome abnormalities. Studies of the sensitivity, specificity, stability and accuracy were performed according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. Compared with the results of the chromosomal microarray analysis (CMA)/copy number variation sequencing (CNV-seq), the detection ratio of non-mosaic chromosome abnormalities of this system in the identification of chromosome 21/18/13/X/Y aneuploidies reached 100%, and the rate of negative results was consistently 100% based on 203 prenatal diagnosis sample analyses. In addition, our results suggested that this panel was a useful tool for mosaicism for Turner syndrome cases. Interestingly, we found one case with large segment loss of chromosome X, which indicated that we should be alert to this situation when the STR genotype of the parent-child is inconsistent in forensic genetics. In summary, this study demonstrated that our system is an accurate, cost-effective and rapid approach for the detection of chromosome numerical abnormalities in prenatal diagnosis.

A novel set of short microhaplotypes based on non-binary SNPs for forensic challenging samples.

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 × 10-22 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.

Genetic reconstruction and phylogenetic analysis by 193 Y-SNPs and 27 Y-STRs in a Chinese Yi ethnic group.

Yi is the seventh-largest ethnic group in China and features mountainous regional characteristics. The Liangshan Yi Autonomous Prefecture is the largest Yi agglomeration with isolated geographical conditions, profoundly impeding genetic communication. Here, we investigated 427 unrelated males of Liangshan from 193 Y-chromosome single nucleotide polymorphisms (Y-SNPs) and 27 Y-chromosome short tandem repeats (Y-STRs) to reveal the genetic structure and paternal phylogeny of the group. The haplogroup diversity reached 0.9169 with 46 different subhaplogroups by 193 Y-SNPs, and the haplotype diversity reached 0.9999 by 27 Y-STR loci. Multidimensional scaling (MDS), N-J tree, and Network were constructed to decipher and visualize the genetic relations between Yi and worldwide groups. Our results revealed: (1) the Network by Y-STRs and Y-SNPs showed the haplogroup D1a1a-M15 in the Liangshan Yi population was a ramification of Tibetan groups' expansion from west to east on the plateau; (2) the haplogroup distribution and the mismatch mutation analysis indicated the haplogroup O2a2b1a1a1a4a2-Z25929 of Liangshan Yi derived from manifold Southeast Asian immigrants; (3) a high-resolution Y-SNPs panel is vital to depict accurate paternal derivations and build an integrated and refining genetic structure of ethnic groups.

An 18 Multi-InDels panel for analysis of highly degraded forensic biological samples.

DNA genotyping from trace and highly degraded biological samples is one of the most significant challenges of forensic DNA identification. There is a lack of simple and effective methods for genotyping highly degraded samples. In this study, a multiple loci insertion/deletion polymorphisms (Multi-InDels) panel was designed for detecting 18 autosomal Multi-InDels through capillary electrophoresis (CE) with amplicon sizes no longer than 125 bp. Studies of sensitivity, degradation, and species specificity were performed and a population study was carried out using 192 samples from Han populations in Hunan province in the south of China. The combined random match probability (CMP) of these 18 Multi-InDels was 3.23 × 10-12 and the cumulative probability of exclusion (CPE) was 0.9989, suggesting this panel could be used independently for human identification and could provide efficient supporting information for parentage testing. Complete profiles were obtained from as low as 62.5 pg of total input DNA after increasing the number of PCR cycles. Moreover, all alleles were detected from artificially highly degraded DNA after 80 min of boiling water bath treatment. This 18 Multi-InDels panel is simple, fast, and effective for the forensic analysis of highly degraded DNA.

Considering the flanking region variants of nonbinary SNP and phenotype-informative SNP to constitute 30 microhaplotype loci for increasing the discriminative ability of forensic applications.

The flanking region variants of nonbinary SNPs and phenotype-informative SNPs (piSNPs) have been observed, which may greatly improve the discriminative ability after constituting microhaplotype. In this study, 30 microhaplotype loci based on the nonbinary SNPs and piSNPs (shown to be related to phenotypes such as hair and eye color) were selected. Genotyping were conducted on 100 unrelated northern Han Chinese, and the 26 populations from the 1000 Genome Project were also included for comparison of populations differentiation. The simulated study was conducted for evaluating the efficiency of kinship testing. These 30 microhaplotype loci we selected had good polymorphism, with a mean effective number of alleles (Ae) of 3.46. The average Ae increase was 1.27 compared with the target SNPs. The populations from the five regions worldwide could also be distinguished using these loci. The results of kinship testing showed that these microhaplotype loci had the similar ability as 15 STR loci of AmpFlSTRR IdentifilerR PCR Amplification Kit to identify the biological parent and a stronger ability to exclude the nonbiological parents. So, these 30 microhaplotype loci may be multifunctional for forensic application, including the ability of personal identification and kinship testing equivalent to 15 STR loci, and the power of ancestry inference for distinguishing the main intercontinental population. Moreover, our selected phenotypic microhaplotype loci may theoretically have phenotype prediction capabilities. But the phenotype prediction efficiency of these phenotypic microhaplotype loci may be worse than that of piSNPs and the detailed prediction accuracy of different populations needs to be further studied.

Exploitation of a novel slowly mutating Y-STRs set and evaluation of slowly mutating Y-STRs plus Y-SNPs typing strategy in forensic genetics and evolutionary research.

The Y-chromosome short tandem repeats (Y-STRs) loci with different mutation rates existing in the Y chromosome non-recombination region (NRY) allow to be applied in human forensics, genealogical researches, historical investigations and evolutionary studies. Currently, there is a high demand for pedigree search to narrow the scope of crime investigations. However, the commonly used Y-STRs kits generally contain Y-STRs with high mutation rates that could cause individuals from the same pedigree to display different haplotypes. Herein, we put forward a new strategy of Slowly Mutating (SM) Y-STRs plus Y-SNPs typing, which could not only improve the resolution and accuracy of pedigree search, but also be applicable to evolutionary research. First, we developed a nine SM Y-STRs assay by evaluating their mutation rates in 210 pedigrees. Then the gene diversity and efficiency of the SM Y-STRs and 172 Y-SNPs sets were investigated by 2304 unrelated males from 24 populations. Furthermore, network and time estimation analyses were performed to evaluate the new strategy's capability to reconstruct phylogenetic tree and reliability to infer the time to the most recent common ancestor (TMRCA). The nine SM Y-STRs assay even had a higher resolution and a comparable capacity of revealing population genetic differentiation compared to 172 Y-SNPs system. This new strategy could optimize the phylogenetic tree generated by commonly used Y-STR panels and obtain a quite consistent time estimations with the published dating.

Forensic characteristics and genetic structure of 18 autosomal STR loci in the Sierra Leone population.

Population genetic analysis is of vital importance for personal identification and paternity testing in forensic science. However, the forensic characteristics of autosomal short tandem repeat (STR) loci in the Sierra Leone population have not been reported yet to the best of our knowledge. In this study, 528 unrelated individuals (256 males and 272 females) in Sierra Leone, West Africa, were genotyped using the DNA Typer19™ kit; forensic parameters and genetic relationships with 32 populations around the world were analyzed. A total of 239 alleles were detected, with corresponding allele frequencies ranged from 0.0009 to 0.4545. The cumulative power of discrimination (CPD) value of the 18 STR loci was 0.9999999999999999999999697; the cumulative probability of exclusion for duos (CPE duos) and exclusion for trios (CPE trios) were 0.99999343 and 0.9999999895, respectively. Genetic comparisons showed that the Sierra Leone population has a closer genetic relationship with the Bantu-speaking populations in Sub-Saharan Africa.

Population data and genetic structure analysis based on 29 Y-STR loci among the ethnolinguistic groups in Burkina Faso.

Burkina Faso (BF) is a landlocked Sahelian country located in the middle of West Africa. Sixty-three local languages are spoken in BF. Despite this high diversity, the BF population remains poorly investigated, and updated forensic parameters with a large number of Y chromosome short tandem repeats (Y-STRs) are still missing. Herein, 447 DNA samples were typed for a cocktail of 29 Y-STR loci. None of these 447 individuals in total shared a common haplotype. The overall Y-STR haplotypes were successfully uploaded online on the Y-STR Haplotype Reference Database (YHRD) with the accession numbers YA004690 and YA004691. The main haplotype diversity was 0.9999999965, which is much higher than that obtained with 12 Y-STRs in a previous study. Haploid Match Probability for the whole dataset was 0.002237. The phylogenetic analysis of 24 ethnic groups of BF shows that the ethnic group named BISSA is closer to Gur speakers than Mande speakers, where they belong. In addition, genetic structure analysis of 49 African subpopulations sheds light on the fact that geographic proximity turns out to be one of the best predictors of genetic affinity between populations.

Developmental validation of the STRscan-17LC kit: a 6 Dye STR kit enhanced stability and ability to detect degraded samples.

Genotyping of short tandem repeat (STR) markers is the basic method of forensic science. Enhanced technologies are needed to meet the requirements of databasing and casework samples. The STRscan-17LC kit is a 6 Dye STR kit which amplifies 16 STR loci: D3S1358, TPOX, D16S539, vWA, D2S1338, CSF1PO, D19S433, D7S820, FGA, D8S1179, D5S818, D13S317, D18S51, TH01, D12S391, and D21S11 and the sex-determinant locus amelogenin. This kit is designed for better tolerance to PCR inhibitors and analysis of mildly degraded samples with all fragments smaller than 330 bases. In this study, the STRscan-17LC kit is validated according to the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines, including PCR-based studies, sensitivity, precision and accuracy, inhibitors, species specificity, DNA mixture studies, population, and concordance studies. The validation results suggest that the STRscan-17LC kit is a useful tool for forensic application.

Forensic parameters and genetic structure analysis of 30 autosomal InDels of the population in Freetown, Sierra Leone.

As the origin of modern humanity, African populations show high genetic diversity and are attracting increasing academic attention. However, populations living in West Africa have so far received less study and exploration. In this study, we analyze 30 insertion/deletion (InDel) loci of 516 samples from Freetown, Sierra Leone, to evaluate the forensic properties and reveal the genetic structure in Freetown, Sierra Leone, West Africa. No significant linkage disequilibrium (LD) between 30 InDels was observed after the Bonferroni correction. The random match probability (RMP), the combined power of exclusion for duos (CPE duos), and the combined power of exclusion for trios (CPE trios) were 6.823 × 10-11, 0.9168, and 0.9731, respectively. Null alleles and off-ladder alleles were observed, suggesting that we should be cautious when using this kit for forensic caseworks in African populations. In the population comparison study, we found that the Freetown population is genetically closer to geographically distinct West Africans and has a closer genetic relationship with the Bantu-speaking populations than other African populations.

Development and validation of novel 8-dye short tandem repeat multiplex system for forensic applications.

DNA profiling of short tandem repeats (STRs) is the primary method for genotyping forensic samples. However, degraded DNA and trace samples are still major problems for commercial 5- or 6-dye STR kits. In order to improve the performance of this method, we developed a novel 8-dye STR multiplex system containing 18 autosomal loci (D3S1358, D1S1656, TPOX, D16S539, vWA, D6S1043, D2S1338, CSF1PO, D19S433, D7S820, FGA, D8S1179, D5S818, D13S317, TH01, D21S11, D12S391, and PentaD) and the sex-determining locus Amelogenin, with all fragments smaller than 330 bases. Validation was carried out as recommended by the Scientific Working Group on DNA Analysis Methods. The results showed that complete profiles were obtainable when the input DNA was as low as 0.0625 ng. Full profiles were obtained even in the presence of inhibitors such as humic acid (< 300 ng/µl), hematin (< 100 µM), and indigo (0.01%). The 8-dye STR multiplex system also showed good performance in the detection degraded DNA samples. These results indicate that the 8-dye STR multiplex system is suitable for human DNA genotyping, including for difficult forensic materials.

Representation of Multiphase OH Oxidation of Amorphous Organic Aerosol for Tropospheric Conditions.

Organic aerosol (OA) is ubiquitous in the atmosphere and, during transport, can experience chemical transformation with consequences for air quality and climate. Prediction of the chemical evolution of OA depends on its reactivity with atmospheric oxidants such as the OH radical. OA particles undergo amorphous phase transitions from liquid to solid (glassy) states in response to temperature changes, which, in turn, will impact its reactivity toward OH oxidation. To improve the predictability of OA reactivity toward OH oxidation, the reactive uptake coefficients (γ) of OH radicals reacting with triacontane and squalane serving as amorphous OA surrogates were measured at temperatures from 213-293 K. γ increases strongest with temperature when the organic species is in the liquid phase, compared to when being in the semisolid or solid phase. The resistor model is applied, accounting for the amorphous phase state changes using the organic species' glass transition temperature and fragility, to evaluate the physicochemical parameters of the temperature dependent OH uptake process. This allows for the derivation of a semiempirical formula, applicable to models, to predict the degree of oxidation and chemical lifetime of the condensed-phase organic species for typical tropospheric temperature and humidity when OA particle viscosity is known.

Validation of the AGCU Expressmarker 16 + 22Y Kit: a new multiplex for forensic application.

Genotyping by targeting short tandem repeats (STRs) has been widely used in forensic applications. However, most commercial kits detect autosomal STRs or Y-STRs alone, which waste both time and opportunity. The AGCU Expressmarker 16 + 22Y Kit includes 16 autosomal and 22 Y-chromosomal STR loci and is designed for the forensic science field and obtaining quicker results. Here, we conducted the validation study according to Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. Validation of PCR-based studies, species specificity, sensitivity, DNA mixture studies, inhibitors, precision, and sizing accuracy were performed. Furthermore, this system was also tested in 346 random male samples from Han, Hui, Tibetan, and Zhuang populations in China, showing its high power for forensic discrimination in the Chinese population. In addition, this system was able to deal with AMELY deletion cases, which can correctly identify sex in forensic criminal investigations. Our results suggested that the AGCU Expressmarker 16 + 22Y Kit is a useful tool for rapid criminal investigation.