Development and application of a multiplex PCR system for forensic salivary identification.

In forensics, accurate identification of the origin of body fluids is essential for reconstructing a crime scene or presenting strong evidence in court. Microorganisms have demonstrated great potential in body fluid identification. We developed a multiplex PCR system for forensic salivary identification, which contains five types of bacteria:Streptococcus salivarius, Neisseria subflava, Streptococcus. mutans, Bacteroides thetaiotaomicron, and Bacteroides. uniformis. And the validated studies were carried out following the validation guidelines for DNA analysis methods developed by the Scientific Working Group on DNA Analysis Methods (SWGDAM), which included tests for sensitivity, species specificity, repeatability, stability, and mixed samples, trace samples, case samples, and a population study. Our result depicted that the lowest detection limit of the system was 0.01 ng template DNA. Moreover, the corresponding bacteria can still be detected when the amount of saliva input is low to 0.1 µL for DNA extraction. In addition, the target bacteria were not detected in the DNA of human, seven common animals, and seven bacteria DNA and in nine other body fluid samples (skin, semen, blood, menstrual blood, nasal mucus, sweat, tears, urine, and vaginal secretions). Six common inhibitors such as indigo, EDTA, hemoglobin, calcium ions, alcohol and humic acid were well tolerated by the system. What is more, the salivary identification system recognized the saliva component in all mixed samples and simulated case samples. Among 400 unrelated individuals from the Chinese Han population analyzed by this novel system, the detection rates of N. subflava, S. salivarius, and S. mutans were 97.75%, 70.75%, and 19.75%, respectively, with 100% identification of saliva. In conclusion, the salivary identification system has good sensitivity, specificity, stability, and accuracy, which can be a new effective tool for saliva identification.

Forensic characteristics and genetic substructure analysis of the Handan Han population, Northern China.

We analysed the forensic characteristics and substructure of the Handan Han population based on 36 Y-STR (short tandem repeat) and Y-SNP (single nucleotide polymorphism) markers. The two most dominant haplogroups in Handan Han, O2a2b1a1a1-F8 (17.95%) and O2a2b1a2a1a (21.51%), and their abundant downstream branches, reflected the strong expansion of the precursor of the Hans in Handan. The present results enrich the forensic database and explore the genetic relationships between Handan Han and other neighbouring and/or linguistically close populations, which suggests that the current concise overview of the Han intricate substructure remains oversimplified.

Dual origins of the Northwest Chinese Kyrgyz: the admixture of Bronze age Siberian and Medieval Niru'un Mongolian Y chromosomes.

The Kyrgyz are a trans-border ethnic group, mainly living in Kyrgyzstan. Previous genetic investigations of Central Asian populations have repeatedly investigated the Central Asian Kyrgyz. However, from the standpoint of human evolution and genetic diversity, Northwest Chinese Kyrgyz is one of the more poorly studied populations. In this study, we analyzed the non-recombining portion of the Y-chromosome from 298 male Kyrgyz samples from Xinjiang Uygur Autonomous Region in northwestern China, using a high-resolution analysis of 108 biallelic markers and 17 or 24 STRs. First, via a Y-SNP-based PCA plot, Northwest Chinese Kyrgyz tended to cluster with other Kyrgyz population and are located in the West Asian and Central Asian group. Second, we found that the Northwest Chinese Kyrgyz display a high proportion of Y-lineage R1a1a1b2a2a-Z2125, related to Bronze Age Siberian, and followed by Y-lineage C2b1a3a1-F3796, related to Medieval Niru'un Mongols, such as Uissun tribe from Kazakhs. In these two dominant lineages, two unique recent descent clusters have been detected via NETWORK analysis, respectively, but they have nearly the same TMRCA ages (about 13th-14th centuries). This finding once again shows that the expansions of Mongol Empire had a striking effect on the Central Asian gene pool.

Y-chromosome evidence confirmed the Kerei-Abakh origin of Aksay Kazakhs.

Aksay Kazakhs are the easternmost branch of Kazakhs, residing in Jiuquan city, the forefront of the ancient Silk Road. However, the genetic diversity of Aksay Kazakhs and its relationships with other Kazakhs still lack attention. To clarify this issue, we analyzed the non-recombining portion of the Y-chromosome from 93 Aksay Kazakhs samples, using a high-resolution analysis of 106 biallelic markers and 17 STRs. The lowest haplogroup diversity (0.38) was observed in Aksay Kazakhs among all studied Kazakh populations. The social and cultural traditions of the Kazakhs shaped their current pattern of genetic variation. Aksay Kazakhs tended to migrate with clans and had limited paternal admixture with neighboring populations. Aksay Kazakhs had the highest frequency (80%) of haplogroup C2b1a3a1-F3796 (previous C3*-Star Cluster) among the investigated Eurasian steppe populations, which was now seen as the genetic marker of Kerei clan. Furthermore, NETWORK analysis indicated that Aksay Kazakhs originated from sub-clan Kerei-Abakh in Kazakhstan with DYS448 = 23. TMRCA estimates of three recent descent clusters detected in C2*-M217 (xM48) network, one of which incorporate nearly all of the C2b1a3a1-F3796 Aksay Kazakhs samples, gave the age range of 976-1405 YA for DC1, 1059-1314 YA for DC2, and 1139-1317 YA for DC3, respectively; this is coherent with the 7th to the 11th centuries Altaic-speaking pastoral nomadic population expansion.

Haplotype analysis of 36 Y-STR loci in a Chinese Han population from Anhui Province, Eastern China.

We analyzed haplotypes for 36 Y chromosomal STRs (Y-STRs), including 27 Yfiler Plus loci and 9 additional STRs (DYS549, DYS643, DYS508, DYS447, DYS596, DYS444, DYS557, and DYS527a/b) in 2018 unrelated Chinese Han individuals from Anhui Province using DNATyperTM 36Y Kit. Phylogenetic analysis was performed to determine the genetic relationship of the Anhui Han population with other neighboring and/or linguistically close populations.

Paternal heritage of the Han Chinese in Henan province (Central China): high diversity and evidence of in situ Neolithic expansions.

Background: Due to their long history, complex admixture processes and large population sizes, more research is required to explore the fine genetic structure of Han populations from different geographic locations of China.Aim: To characterise the paternal genetic structure of the Han Chinese in Henan province, which was once the central living region of the ancient Huaxia population, the precursors of the Han Chinese.Subjects and methods: We sequenced Y chromosomes of 60 males from Zhengzhou, Henan Province, and reconstructed a phylogenetic tree for these samples with age estimation.Results: We observed high diversity of paternal lineages in our collection. We found that the in situ Neolithic expansion of the "Major lineages" contributed to a large portion of the paternal gene pool of the Han population in Henan Province. We also detected a large number of "Minor lineages" that diverged in the Palaeolithic Age.Conclusion: We suggest that the high genetic diversity in the paternal gene pool of modern Han populations is mainly attributed to the reservation of a larger number of lineages that diverged in the Palaeolithic Age, while the recent expansion of limited lineages contributed to the majority of the gene pool of modern Han populations. We propose that such a structure is a basal characteristic for the genetic structure of modern Han populations.

Relating Clans Ao and Aisin Gioro from northeast China by whole Y-chromosome sequencing.

The Y-chromosome haplogroup C2b1a3a2-F8951 is the paternal lineage of the Aisin Gioro clan, the most important brother branch of the famous Mongolic-speaking population characteristic haplogroup C2*-Star Cluster (C2b1a3a1-F3796). However, investigations on its internal phylogeny are still limited. In this study, we used whole Y-chromosome sequencing to update its phylogenetic tree. In the revised tree, C2b1a3a2-F8951 and C2*-Star Cluster differentiated 3852 years ago (95% CI = 3295-4497). Approximately 3558 years ago (95% CI = 3013-4144), C2b1a3a2-F8951 was divided into two main subclades, C2b1a3a2a-F14753 and C2b1a3a2b-F5483. Currently, samples of C2b1a3a2-F8951 were mainly from the House of Aisin Gioro clan, the Ao family from Daur and some individuals mainly from northeast China. Although other haplogroups are also found in the Ao family, including C2b1a2-M48, C2b1a3a1-F3796, C2a1b-F845, and N1c-M178, the haplogroup C2b1a3a2-F8951 is still the most distinct genetic component. For haplogroup C2b1a3a2-F8951, the time of the most recent common ancestor of the House of Aisin Gioro clan and the Ao family were both very late, just a few hundred years ago. Some family-specific Y-SNPs of the House of Aisin Gioro and the Ao family were also discovered. This revision evidently improved the resolving power of Y-chromosome phylogeny in northeast Asia, deepening our understanding of the origin of these two families, even the Mongolic-speaking population.

Molecular genealogy of Tusi Lu's family reveals their paternal relationship with Jochi, Genghis Khan's eldest son.

Genghis Khan's lineage has attracted both academic and general interest because of its mystery and large influence. However, the truth behind the mystery is complicated and continues to confound the scientific study. In this study, we surveyed the molecular genealogy of Northwestern China's Lu clan who claim to be the descendants of the sixth son of Genghis Khan, Toghan. We also investigated living members of the Huo and Tuo clans, who, according to oral tradition, were close male relatives of Lu clan. Using network analysis, we found that the Y-chromosomal haplotypes of Lu clan mainly belong to haplogroup C2b1a1b1-F1756, widely prevalent in Altaic-speaking populations, and are closely related to the Tore clan from Kazakhstan, who claim to be the descendants of the first son of Genghis Khan, Jochi. The most recent common ancestor of the special haplotype cluster that includes the Lu clan and Tore clan lived about 1000 years ago (YA), while the Huo and Tuo clans do not share any Y lineages with the Lu clan. In addition to the reported lineages, such as C3*-Star Cluster, R1b-M343, and Q, our results indicate that haplogroup C2b1a1b1-F1756 might be another candidate of the true Y lineage of Genghis Khan.

Population genetic analysis of the Globalfiler STR loci in 3032 individuals from the Altay Han population of Xinjiang in northwest China.

The genetic polymorphisms of 21 autosomal short tandem repeat (STR) loci included in the GlobalFiler™ PCR Amplification Kit were evaluated in 3032 unrelated individuals Altay Han of Xinjiang, northwest China. All of the loci reached the Hardy-Weinberg equilibrium. These loci were examined to determine allele frequencies and forensic statistical parameters. SE33 showed the greatest power of discrimination in Altay Han population, whereas TPOX showed the lowest. The combined discrimination power and probability of excluding paternity of the 21 autosomal STR loci were 0.999999999999999999999999889838 and 0.999999996664704, respectively. Both pairwise genetic distance and phylogenetic methods indicated that the Altay Han had the closest genetic relationship with the Han origin and Hui populations. The present results revealed that the GlobalFiler system had a high level of polymorphism in Altay Han population and hence could be a powerful tool for forensic application and population genetic study.

Revisiting the male genetic landscape of China: a multi-center study of almost 38,000 Y-STR haplotypes.

China has repeatedly been the subject of genetic studies to elucidate its prehistoric and historic demography. While some studies reported a genetic distinction between Northern and Southern Han Chinese, others showed a more clinal picture of small differences within China. Here, we investigated the distribution of Y chromosome variation along administrative as well as ethnic divisions in the mainland territory of the People's Republic of China, including 28 administrative regions and 19 recognized Chinese nationalities, to assess the impact of recent demographic processes. To this end, we analyzed 37,994 Y chromosomal 17-marker haplotype profiles from the YHRD database with respect to forensic diversity measures and genetic distance between groups defined by administrative boundaries and ethnic origin. We observed high diversity throughout all Chinese provinces and ethnicities. Some ethnicities, including most prominently Kazakhs and Tibetans, showed significant genetic differentiation from the Han and other groups. However, differences between provinces were, except for those located on the Tibetan plateau, less pronounced. This discrepancy is explicable by the sizeable presence of Han speakers, who showed high genetic homogeneity all across China, in nearly all studied provinces. Furthermore, we observed a continuous genetic North-South gradient in the Han, confirming previous reports of a clinal distribution of Y chromosome variation and being in notable concordance with the previously observed spatial distribution of autosomal variation. Our findings shed light on the demographic changes in China accrued by a fast-growing and increasingly mobile population.

Analysis of 27 Y-chromosomal STR haplotypes in a Han population of Henan province, Central China.

A total of 1225 unrelated Han males from Henan province were analyzed with the prototype Yfiler® Plus kit (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). The calculated gene diversity (GD) values ranged from 0.3855 to 0.9673 for the DYS391 and DYS385a/b loci, respectively. The discriminatory capacity (DC) was 86.94 % with 1065 observed haplotypes using 17 Yfiler loci, by the addition of 10 Y-STRs to the Yfiler® Plus system, the DC was increased to 98.94 % while showing 1212 observed haplotypes. Among the new incorporated Y-STRs, DYS576, DYF387S1, DYS518, DYS627, and DYS449 were major contributors to enhancing discrimination. In the analysis of molecular variance, the Henan Han population clustered with Asian origin populations and showed significant differences from other reference populations. In this study, the improvement of adding additional Y-STR markers with the Yfiler® Plus kit provided substantially stronger discriminatory power in the Henan Han population.

[Application of Multiple Genetic Markers in a Case of Determination of Half Sibling].

OBJECTIVE: A case of half sibling was determined with multiple genetic markers, which could be potentially applied for determination of half sibling relationship from same father. METHODS: Half sibling relationship was detected by 39 autosomal STR genetic markers, 23 Y-chromosomal STR genetic markers and 12 X -chromosomal STR genetic markers among ZHAO -1, ZHAO -2, ZHAO -3, ZHAO -4, and ZHAO-5. RESULTS: According to autosomal STR, Y-STR and X-STR genotyping results, it was determined that ZHAO-4 (alleged half sibling) was unrelated with ZHAO-1 and ZHAO-2; however, ZHAO-3 (alleged half sibling), ZHAO-5 (alleged half sibling) shared same genetic profile with ZHAO-1, and ZHAO-2 from same father. CONCLUSION: It is reliable to use multiple genetic markers and family gene reconstruction to determine half sibling relationship from same father, but it is difficult to determination by calculating half sibling index with ITO and discriminant functions.

[Establishment and Verification of 6-color Fluorescent-labeled Rapid PCR Amplification System].

OBJECTIVE: To establish the rapid PCR amplification program and system and to verify the technical indexes. METHODS: PCR multiplex and capillary electrophoresis detection of 24 autosomal STR loci and one Y-STR loci using the 6-color fluorescence marking technology, as well as A melogenin and Y-InDel. Meanwhile, sensitivity, specificity, identity, stability, mixing and a batch of sample tests were investigated, and the genotype of various routine samples and degraded, exfoliated cell samples were observed. RESULTS: The sensitivity of the system was 0.062 5 ng. In addition, the genotype could be detected accurately only around 65 min via rapid amplification. The species-specificity was high and the genotyping of all kinds of dry blood specimens of filter paper and mixed, degraded, exfoliated cell samples were accurate. CONCLUSION: The rapid amplification system can significantly improve the detection rate, and obtain accurate and stable genotyping results, which may be important implications for the establishment of STR database and study on population genetics and forensic identification.

Population data of 30 insertion-deletion markers in four Chinese populations.

In this study, we assessed 30 insertion-deletion polymorphisms (Indels) (Investigator DIPplex® kit) in four Chinese populations (n = 952) and evaluated their usefulness in forensic genetic applications. After the Bonferroni correction at a 95 % significance level (p = 0.0017), there were no deviations from the Hardy-Weinberg equilibrium observed except for the HLD114 locus in the Tibetan ethnic group studied. A high level of discrimination power was observed for the DIPplex® kit in four sample populations (CDP > 0.9999) and the combined random match probabilities (CMP) were in the range of 1.4766 × 10(-11) to 5.2742 × 10(-13). Four Indels have been selected for further analyses as possible ancestry informative single nucleotide polymorphisms. The study support that the Investigator DIPplex® kit provides a powerful supplement to standard short tandem repeat-based kits for individual identification and kinship analysis in the Chinese population.

[Genetic polymorphisms of 30 InDel loci in Chinese ethnic population residing in Tibet].

OBJECTIVE: To analyze the genetic data of 30 insertion and deletion polymorphisms (InDel) loci included in an InvestigatorR DIPplex diagnostic kit, and to evaluate the forensic application in ethnic Tibetan population from China. METHODS: By detecting 226 unrelated individuals with the Investigator(R) DIPplex kit, allelic frequencies and population genetics parameters of the 30 InDels were statistically analyzed and compared with available data derived from other populations from various regions. RESULTS: After the Bonferroni correction at a 95% significance level (P=0.0017), no significant departures from the Hardy-Weinberg equilibrium were observed except for the HLD114 locus. Linkage disequilibrium test showed no significant allelic association between all 30 loci after the Bonferroni's correction. The average heterozygosity (Ho) of all loci was 0.4125, the mean discrimination power (DP) was 0.5618, the mean polymorphism information content (PIC) was 0.3280, and the combined discrimination power (TDP) was 0.999999999990. The combined power of exclusion of all loci was 0.987 849 91 in trio cases and 0.94977125 in duo cases. Genetic distance between Tibetan and Han from Beijing was minimum (0.0068) in the 5 populations, while genetic distance between Tibetan and Uygur was maximal (0.0215). CONCLUSION: Multiplex detection has revealed that these 30 InDel loci have a moderate distribution of genetic polymorphism among ethnic Tibetan group residing in Tibet, China.

Forensic discrimination of three common brands of kitchen knives in China by ICP-AES and infrared absorption.

A block of an injury instrument will be left in wounds sometimes, and the suspect instrument can be discriminated by comparison with the block that was left through elemental analysis. In this study, three brands (Shibazi, Zhangxiaoquan, Qiaoxifu) of kitchen knives with forged, chop, and slice application series were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and Infrared Absorption to investigate the type, number of elements and the reference range used for comparing. The results show that when regarding one or more element as the discriminative threshold, together with 5% relative standard deviation (RSD) as the reference range, all the samples could be distinguished among different series. Furthermore, within the same series, the discriminative capability could reach up to 88.57% for all samples. In addition, elements with high content, such as Cr, Mn, and C, were useful to discriminate among different series, and trace elements, such as Ni, Si, and Cu, were useful within the same series. However, in practice, it is necessary to evaluate the accuracy of the method by Standard Reference Material (SRM) before an examination is performed.

[Analysis of linkage disequilibrium and linkage for 12 short tandem repeat loci on chromosome X].

OBJECTIVE: To analyze linkage disequilibrium of 12 short tandem repeat loci on chromosome X (X-STR) among an ethnic Han population from Guilin, Guangxi, and to study the genetic linkage and haplotype distributions of such loci in 2 linkage groups. METHODS: 12 X-STR loci including DXS8378, DXS10159, DXS10162, DXS10164, DXS981, DXS6789, DXS7424, DXS101, DXS7133, GATA165B12, GATA31E08 and DXS7423 were genotyped using an AGCU X12 STR PCR Amplification kit. A total of 119 pedigrees were analyzed for linkage and linkage disequilibrium. RESULTS: Two mutations were found at DXS7424, and 1 mutation was found at DXS10164. A total of 93 haplotypes of DXS10159-DXS10162-DXS10164 were constructed for 261 unrelated males and females, in addition with 167 haplotypes of DXS6789-DXS7424-DXS101-DXS7133. The values of recombination fraction between DXS10159 and DXS10162, DXS10162 and DXS10164, DXS6789 and DXS7424, and DXS7424 and DXS101 were 0.0269, 0.0236, 0.0505 and 0.0438, respectively. CONCLUSION: Linkage disequilibrium of X-STR does not only depend on physical and genetic distances. There was incomplete linkage relationship between loci on DXS10159-DXS1016-DXS10164 and DXS6789-DXS7424-DXS101 linkage groups.

Forensic analysis and sequence variation of 133 STRs in the Hakka population.

Introduction: Short Tandem Repeats (STRs) are highly valuable genetic markers in forensic science. However, the conventional PCR-CE technique has limitations, and the emergence of massively parallel sequencing (MPS) technology presents new opportunities for STR analysis. Yet, there is limited research on Chinese population diversity using MPS. Methods: In this study, we obtained genotype data for 52 A-STRs and 81 Y-STRs from the Hakka population in Meizhou, Guangdong, China, using the Forensic Analysis System Multiplecues SetB Kit on the MGISEQ-2000 platform. Results: Our findings demonstrate that these 133 STRs are highly efficient for forensic applications within the Meizhou Hakka population. Statistical analysis revealed Hobs values ranging from 0.61306 to 0.91083 and Hexp values ranging from 0.59156 to 0.91497 for A-STRs based on length polymorphism. For sequence polymorphism, Hobs values ranged from 0.61306 to 0.94586, and Hexp values fluctuated between 0.59156 and 0.94487. The CPE values were 1-5.0779620E-21 and 1-3.257436E-24 for length and sequence polymorphism, respectively, while the CPD values were 1-1.727007E-59 and 1-5.517015E-66, respectively. Among the 80 Y-STR loci, the HD values for length and sequence polymorphism were 0.99764282 and 0.99894195, respectively. The HMP values stood at 0.00418102 and 0.00288427, respectively, and the DC values were 0.75502742 and 0.83363803, respectively. For the 52 A-STR loci, we identified 554 and 989 distinct alleles based on length and sequence polymorphisms, respectively. For the 81 Y-STR loci, 464 and 652 unique alleles were detected at the length and sequence level, respectively. Population genetic analysis revealed that the Meizhou Hakka population has a close kinship relationship with the Asian populations THI and KOR based on length polymorphism data of A-STRs. Conversely, based on length polymorphism data of Y-STRs, the Meizhou Hakka population has the closest kinship relationship with the Henan Han population. Discussion: Overall, the variation information of repeat region sequences significantly enhances the forensic identification efficacy of STR genetic markers, providing an essential database for forensic individual and paternity testing in this region. Additionally, the data generated by our study will serve as a vital resource for research into the genetic structure and historical origins of the Meizhou Hakka population.

Systematic analyses of AISNPs screening and classification algorithms based on genome-wide data for forensic biogeographic ancestry inference.

Identifying the biogeographic ancestral origin of biological sample left at a crime scene can provide important evidence for judicial case, as well as clue for narrowing down suspect. Ancestry informative single nucleotide polymorphism (AISNP) has become one of the most important genetic markers in recent years for screening ancestry information loci and analyzing the population genetic background and structure due to their high number and wide distributions in the human genome. In this study, based on data from 26 populations in the 1000 Genomes Project Phase 3, a Random Forest classification model was constructed with one-vs-rest classification strategy for embedded feature selection in order to obtain a panel with a small number of efficient AISNPs. The research aim was to clarify differentiations of population genetic structures among continents and subregions of East Asia. ADMIXTURE results showed that based on the 58 AISNPs selected by the machine learning algorithm, the 26 populations involved in the study could be categorized into six intercontinental ancestry components: North East Asia, South East Asia, Africa, Europe, South Asia, and America. The 24 continental-specific AISNPs and 34 East Asian-specific AISNPs were finally obtained, and used to construct the ancestry prediction model using XGBoost algorithm, resulting in the Matthews correlation coefficients of 0.94 and 0.89, and accuracies of 0.94 and 0.92, respectively. The machine learning models that we constructed using population-specific AISNPs were able to accurately predict the ancestral origins of continental and intra-East Asian populations. To summarize, screening a set of high-perform AISNPs to infer biogeographical ancestral information using embedded feature selection has potential application in creating a layered inference system that accurately differentiates from intercontinental populations to local subpopulations.