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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.

Y-chromosome short tandem repeat (Y-STR) and Y-chromosome single nucleotide polymorphism (Y-SNP) frequency distributions provide resources for assessment of male population stratification among world-wide populations. Currently, the Y-STR Haplotype Reference Database (YHRD) contains numerous Y-chromosome haplotype profiles from various populations and countries around the world. However, for many of the recently discovered and already phylogenetically mapped Y-SNPs, the population data are scarce. Herein, the typing of 27 Y-STRs (Yfiler Plus) and 143 Y-SNPs (self-designed Y-SNP panel) was performed on 1269 unrelated males from 11 Han Chinese populations. Haplogroup O-M175 was the most predominant haplogroup in our Han Chinese data, ranging from 67.34% (Henan Han) to 93.16% (Guangdong Han). The highest haplogroup diversity (0.967056) was observed in Heilongjiang Han, with a discrimination capacity (DC) value of 0.3723. The number of alleles at single-copy loci varied from 2 for DYS391 (Guangdong Han) to 16 for DYS518 (Henan Han). For the majority of the populations (8/11), both the haplotype diversity and DC values are 1.0000. Furthermore, genetic differentiations were observed between Northern and Southern Han Chinese. These genetic differences were mainly reflected in haplogroup distribution and frequency, and they were confirmed by statistical analysis.

During the last decade, RNA profiling has emerged as one of the fastest developed methods for discriminating forensically relevant biological samples. As a category of small non-coding RNA, piwi-interacting RNA (piRNA) has recently been proposed to be differentially expressed in different types of body fluids, which indicates that its potential in forensic science is worth exploring. In this study, small RNA from 6 types of biological samples (venous blood, menstrual blood, saliva, semen, vaginal secretions and skin) was prepared and sequenced in order to characterize the expression pattern of piRNA using Ion S5 XL platform. Multiple bioinformatic methods were applied to make interpretation of the massively parallel sequencing data and identify representative biomarkers. A total of 376 piRNAs were initially identified after normalization and filtering. Hierarchical clustering and partial least squares-discriminant analysis (PLS-DA) revealed that their expression profiles exhibited an acceptable discriminating ability for most biological samples. Besides, a panel consists of 37 piRNA candidates was subsequently established for further analysis. The results suggested that with the optimal number of PLS components, the marker-reduced panel was sufficient to construct a PLS-DA model with the same performance as that can be achieved by the entire 376 piRNAs (classification error rate = 0.04). In addition, 5 targeted candidates were further selected for validation. TaqMan RT-qPCR assay results verified the potential of 3 piRNAs (piR-hsa-27622, piR-hsa-1207 and piR-hsa-27493) in distinguishing venous blood and menstrual blood, as well as 2 piRNA (piR-hsa-27493 and piR-hsa-26591) for the discrimination of saliva and vaginal secretions, which emphasized the feasibility of our biomarker selection approach. In brief, our study expanded the amount of potential piRNA biomarkers and demonstrated that the expression features of piRNA could provide valuable information for discriminating forensically relevant biological samples.

Y-Chromosomal short tandem repeat polymorphisms (Y-STRs) are widely applied in human forensic cases and population genetic studies. There is a lack of information about the Sichuan Tibetan population in the Y-STR Haplotype Reference Database (YHRD, https://yhrd.org, release 59). In this study, 502 unrelated male individuals residing in the Sichuan Province were recruited and genotyped at 29 Y-STR loci. A total of 479 haplotypes were observed, 460 (96.03%) of which were unique. The haplotype diversity (HD) and discrimination capacity (DC) for the Sichuan Tibetan population were 0.9998 and 0.9542, respectively. To reveal the genetic diversities and relationships between the Chinese Sichuan Tibetan and 29 other previously reported populations, forensic parameter analysis, multi-dimensional scaling, and phylogenetic reconstruction were performed. The results showed that the Sichuan Tibetan population was relatively isolated from other populations, suggesting that genetic proximity is in line with geographical boundaries.

Massively parallel sequencing (MPS) technology has the capability to genotype hundreds to thousands of SNPs from multiple samples, with high coverage in a single experimental run. MPS, which enables high-resolution Y haplogrouping from limited DNA resources, has attracted much interest in Y-chromosomal applications, particularly human forensics and anthropological studies. Previously, we selected and designed a 72 Y-SNPs MPS panel aiming at the Chinese population for providing the basic structure of the haplogroup tree, and then additional Y-SNPs were screened out for further subdivision into different populations by using pyrosequencing technology. In the present study, we designed a customized MPS panel that includes 165 Y-SNPs based on our previous studies and evaluated the sequencing performance, sensitivity and the ability of the MPS-SNP panel to analyze case-type samples using the Ion S5 XL System. In the concordant study, only four Y-SNPs presented missing genotypes due to low coverage reads, and five SNPs were prone to being designated heterozygotes, whereas the designation of the other SNPs were fully consistent with the results obtained from other Y-SNP typing tools. The analyst-interpreted Y-SNP profiles demonstrated that the targeted Y-SNPs, except for the nine poorly performing Y-SNPs, performed well and could acquire achievable high-resolution. Moreover, this MPS panel could be adapted to case-type samples, and single source complete profiles could be stably obtained using 1 ng of input DNA. In summary, this MPS Y-SNP panel offers a straightforward sample-to-haplogroup workflow that would be beneficial for paternal lineage classification and forensic pedigree searches, and the present results provide support for the application of MPS technology in SNP analysis.

The X chromosome has a special mode of inheritance, and is thus a rich resource for population studies. In this study, the allele frequencies and forensic statistics of the 19 X chromosomal short tandem repeat loci were evaluated in 500 Uyghur individuals from Aksu Prefecture in northwest China. We further aimed to study whether the Uyghur populations located in various regions of Xinjiang share similar allele and haplotype frequency distributions, as they have experienced genetic exchanges. Population comparisons, PCA and MDS were performed for the Uyghurs and 27 populations and the results indicate that the Uyghur minority in Aksu has a relatively close phylogenetic relationship with East Asians, especially the Kazakh minority.

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.

With the widespread use of STR in identification of individuals, paternity testing, as well as population genetics, many commercially robust and validated STR multiplex kits were developed. The AGCU Expressmarker 30 Kit is a new autosomal STR system that contains 29 autosomal STR loci (D3S1358, vWA, D1S1656, CSF1PO, D8S1132, D19S253, D3S3045, D8S1179, D21S11, D16S539, TPOX, D6S477, Penta D, D2S441, D5S818, TH01, FGA, D15S659, D22S1045, D19S433, D13S317, D7S820, D6S1043, D10S1435, D10S1248, D2S1338, D18S51, D12S391, and Penta E), one insertion/deletion polymorphic marker on the Y chromosome (Y indel), and the amelogenin locus. A series of validation studies were performed in this context according to the guidelines of “Validation Guidelines for Forensic DNA Analysis Methods”. The sensitivity study showed that a full profile was observed with template DNA as low as 40 pg. In the stability study, all STR profiles were obtained at concentrations of humic acid up to 800 ng/μL, hematin up to 250 μM, and tannic acid up to 200 ng/μL. The mixture study demonstrated that all of the minor alleles could be called at ratios from 1:1–29:1 when the total DNA was 2 ng. In the population study, the total discrimination power for three population (Sichuan-Han, Gansu-Hui, and Guangxi-Zhuang) were above 0.9999999999999999999999999999999992, 0.999999999999999999999999999999998 and 0.999999999999999999999999999999994 as well as the cumulative probability of paternity exclusion were 0.999999999999953, 0.999999999999178, and 0.999999999999611 respectively. These results demonstrated that the AGCU Expressmarker 30 Kit is a useful tool for analyzing both forensic casework and database samples.

The Hui group is the second largest ethnic minority and one of the most widespread ethnic groups in China. However, the genetic architecture of the Hui population remains largely unexplored, particularly with respect to the male-specific region of the Y chromosome. Here, we studied nine Hui populations (Xinjiang, Qinghai, Gansu, Ningxia, Shaanxi, Henan, Shandong, Sichuan, Yunnan) using 157 Y-chromosome single nucleotide polymorphisms (Y-SNPs) and 27 short tandem repeats (Y-STRs) to unravel their genetic substructure and forensic characteristics. A total of 650 unrelated male samples from the Hui populations were genotyped by SNaPshot®, a single base extension (SBE) assay. Finally, 95 terminal haplogroups and high haplotype diversity (0.9999) were observed in Hui populations. Frequency heat map matrices, genetic distance (FST) and network analysis within Hui populations indicated that these nine Hui populations can be divided into three groups: Hui populations from the northwest (NWH), Hui populations from Sichuan and Shandong (SSH), and Hui populations from Yunnan (YNH). Our results suggested that we should use different databases for different Hui samples in forensic cases. Comparison with other populations that used different population genetic analysis revealed that the Hui populations had close relationships with East Asian populations, especially Chinese Han population. Overall, the high-resolution panel with Y-SNPs and Y-STRs gives new and complete insight into Hui populations, which can be used to interpret the genetic substructure of Hui populations and affect the utility of forensic databases.

Hainan Island is the southernmost and smallest Chinese province, isolated from the mainland. The Li and Han ethnic groups account for over 98% of the population on the island. However, the Li ethnic group is an indigenous community of Hainan Island, with great differences in culture, language and origin with respect to the Han, the largest ethnic group. Here, we studied these two ethnic groups from the perspective of the Y-chromosome single nucleotide polymorphisms (Y-SNPs) and short tandem repeats (Y-STRs) to unravel their forensic and phylogenetic characteristics. A total of 302 unrelated male samples from the Li and Han ethnic groups in Hainan Island were genotyped by a combination of three separate typing systems (next-generation sequencing and pyrosequencing for the Y-SNPs and capillary electrophoresis for the Y-STRs), a previously developed high-resolution panel containing 141 Y-SNPs and 27 Y-STRs. The haplotype diversity of the Li ethnic group reached 0.9997, and 49 terminal haplogroups were observed in the Li and Han ethnic groups. Haplogroup O1b1a1a1a1a1b-CTS5854 was the most predominant haplogroup, including 44.12% of Li individuals. Median-joining trees showed little gene flow between the Li and Han individuals, as well as between the Li and other ethnic groups in Hainan Island. Our results indicated that 1) in contrast with the Han ethnic group, a low degree of genetic diversity was observed in the Li ethnic group; 2) there was limited gene flow between the Li and Han ethnic groups; and 3) founder effect was identified in the Li ethnic group in Hainan Island.

In crime scenes, age prediction of biological stains could be of great significance in searching the suspects and reconstructing the events. Age-related CpGs (AR-CpGs) has been increasingly recognized because of its potential application for forensic age prediction. Currently, the MethBank database with 34 consensus reference methylomes from 4577 human blood samples was available. The current study aimed to develop a strategy for screening out the AR-CpGs markers based on both MethBank database and the pyrosequencing methods. Four AR-CpGs were selected for DNA methylation analysis in Chinese Han population. Twenty blood stains which were stored for over one month were detected by pyrosequencing approach. The results implied that four AR-CpGs may be potential markers for age estimating.

In forensic science, the reliable identification of human body fluids could provide important information for crime investigation. Circular RNAs (circRNAs), with an abundant, stable and tissue/developmental-stage-specific expression, may be as potential markers for forensic body fluid identification. In this study, microarray expression profiles of circRNAs were explored with total RNA extraction from venous blood, semen, saliva, vaginal secretion, and menstrual blood samples. As results, semen, saliva and venous blood could be easily distinguished from each other based on the circRNAs expression profiles, but vaginal secretions and menstrual blood displayed similar signatures. This study suggested that circRNAs extraction from forensic samples was possible and the microarray screening of circRNAs could enrich the candidates for forensic body fluid identification.