Forensic Characterization and Genetic Portrait of the Gannan Tibetan Ethnic Group via 165 AI-SNP Loci

Background : The Tibetan group is one of the oldest Sino-Tibetan ethnic groups. The origin, migration as well as the genetic background of Tibetans have become the research hotspots in the field of forensic genetics. The use of ancestry informative markers (AIMs) allows the investigation of the genetic background of the Gannan Tibetan group. Methods : In this study, the 165 ancestry informative single nucleotide polymorphism (AI-SNP) loci included in the Precision ID Ancestry Panel were used to genotype 101 Gannan Tibetans using the Ion S5 XL system. The forensic statistical parameters of 165 AI-SNP in the Gannan Tibetan group were calculated. Population genetic analyses including Nei’s genetic distances, phylogenetic analyses, pairwise fixation index, principal component analyses and population ancestry composition analyses were also conducted to evaluate the genetic relationships between the Gannan Tibetan group and other reference populations. Results : Forensic parameters of the 165 AI-SNP loci indicated that not all of the SNPs showed high genetic polymorphisms in the Gannan Tibetan group. Population genetic analyses indicated that the Gannan Tibetan group had close genetic affinities with East Asian populations, especially with the groups residing in its neighboring geographical regions. Conclusions : The 165 AI-SNP loci in the Precision ID Ancestry Panel showed high ancestral prediction powers for different continental populations. When trying to predict the ancestral information of East Asian subpopulations using this panel, the prediction results are not particularly accurate. The 165 AI-SNP loci showed varying degrees of genetic polymorphisms in the Gannan Tibetan group, and the combined use of these loci could be an effective tool in the forensic individual identification and parentage testing of this group. The Gannan Tibetan group has close genetic affinities with East Asian populations compared with other reference populations, especially tighter genetic relationships with the groups residing in its neighboring geographical regions.


Introduction
The Tibetan group, indigenous group of the Qinghai-Tibet Plateau, is one of the 56 ethnic groups with a long history in China. Chinese Tibetan group mainly settled in the Tibet Autonomous Region, Qinghai, Western Sichuan, Yunnan, and the Gansu provinces [1,2]. According to the seventh national population census in 2020, the population of the Tibetans is more than seven millions [3]. The Tibetan language is a branch of the Tibeto-Burman family of the Sino-Tibetan language. Chinese Tibetan group could be divided into three main subgroups (U-Tsang, Khams and Amdo) in terms of their geographical and cultural differences [4]. The Gansu Tibetans, belonging to a branch of the Amdo Tibetans, mainly reside in Gannan Tibetan Au-tonomous Prefecture, which is located on the northeastern edge of the Qinghai-Tibet Plateau, and at the border of the Aba Tibetan Qiang Autonomous Prefecture of the Sichuan province [5]. In the past decade, many researchers devoted their efforts to uncovering the origin, migration, genetic admixture as well as high-altitude adaptation of the Tibetan group [1,[6][7][8]. However, these issues have been long-standing heated arguments because of limited genetic data of ancient and modern Tibetans.
In recent years, ancestry inference of unknown DNA donor found at the crime scene has become a new tool to help solve crime [9]. Ancestry informative marker (AIM) refers to a kind of genetic markers with significant frequency variations among different populations. With the technological developments of next generation sequencing (NGS) and the reduction of the sequencing cost, several biogeographical ancestry inference panels based on single nucleotide polymorphism (SNP), deletion-insertion polymorphism (DIP) and multi-allelic haplotype markers have been constructed [10][11][12]. Nowadays, ancestry information inference has wide applications not only in crime investigations, but also in the identifications of skeletal remains of missing persons, population substructure studies, population genetic investigations, and the disease susceptibility studies in different populations [13][14][15][16].
With the exception of AI-SNP panels recently developed for East Asian subpopulation structure [17][18][19][20][21], few commercial kits are available in the forensic genetic field. ForenSeq™ DNA Signature Prep Kit and Precision ID Ancestry Panel are both commonly used in forensic DNA laboratory. According to related validation study, the 165 AI-SNP loci of the Precision ID Ancestry Panel can be used to discriminate the ancestal origins of major populations including Africa, Europe, Southwest Asia, South Asia, East Asia, Oceania and Americas [22]. Population genetic evaluations based on Precision ID Ancestry Panel indicated that this panel could also be used to analyze the genetic background of some subpopulations [23,24]. In this study, we systematically evaluated the performance of the Precision ID Ancestry Panel in 101 Tibetan individuals from Gannan Tibetan Autonomous Prefecture of Chinese Gansu province. The present results provided the raw data on genetic variations at 165 SNP loci in Gannan Tibetan ethnic minority, the relevant forensic parameters for individual discrimination in this region and indicated the genetic relationships between Gannan Tibetan group and reference groups.

Ethical Statement and Sample Collection
The present study has been approved by the ethics committee of Xi'an Jiaotong University Health Science Center and Southern Medical University (Ethical Approval Number: 2019-1039). This research was conducted in accordance with the ethical principle for medical research involving human subjects recommended by the World Medical Association Declaration of Helsinki. Sample collection and the following sequencing experiments were performed in strict compliance with the ethical regulations. Peripheral venous blood samples were collected from 101 unrelated volunteers who lived in the Gannan Tibetan Autonomous Prefecture in the Gansu province, China for at least three generations. The blood samples were dried on the FTA cards, and then stored at room temperature. Each volunteer signed an informed consent form before the sample collection.

Genomic DNA Extraction and DNA Quantification
For each sample, five pieces of 1.0 mm 2 bloodstain were used to extract the genomic DNA using the QIAamp® DNA Investigator kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions [25]. The concentration of genomic DNA was then measured by quantitative fluorescence using the Qubit™ dsDNA HS Assay kit (Thermo Fisher Scientific Inc, Waltham, MA, USA) and the Qubit 4.0 Fluorometer (Thermo Fisher Scientific Inc, Waltham, MA, USA) following the manufacturer's recommendations [26].

Library Preparation and Quantification
The Precision ID DL8 Kit and the Precision ID Ancestry Panel (Thermo Fisher Scientific Inc, Waltham, MA, USA) were used to prepare DNA library on the automated Ion Chef™ system (Thermo Fisher Scientific Inc, Waltham, MA, USA). For each sample, 15 µL of DNA (1 ng in total) was pipetted into A1 to A8 wells of a Precision ID DL8 IonCode™ Barcode Adapters plate. And twenty-one cycles were used to amplify the target regions. The concentrations of sample libraries were measured by the Ion Library TaqMan® Quantitation Kit (Thermo Fisher Scientific Inc, Waltham, MA, USA) on the QuantStudio™ 5 realtime PCR system (Thermo Fisher Scientific Inc, Waltham, MA, USA) following the manufacturer's instructions. Before template preparation, all DNA libraries were pooled in equimolar concentrations (33 pM).

Template Preparation and DNA Sequencing
Template preparation, enrichment of beads with template, and the sequence of template on beads were performed with the Ion Chef™ and Ion S5™ XL (Thermo Fisher Scientific Inc, Waltham, MA, USA) instruments according to the manufacturer's instructions. After template preparation, high-throughput sequencing was preformed using four pieces of Ion™ 530 chips (Thermo Fisher Scientific Inc, Waltham, MA, USA). Thirty-two samples were loaded on each Ion™ 530 chip. Positive DNA 007 and deionized water were used as positive and negative controls in each run.

Analyses of NGS Data
The analysis of primary sequence data was performed with the Torrent Suite Software (Thermo Fisher Scientific Inc, Waltham, MA, USA). All reads were aligned to hg19 reference sequences. Further sequence analyses were carried out using the HID-SNP Genotyper v4.2 plugin and Converge™ Software (Thermo Fisher Scientific Inc, Waltham, MA, USA) with default analysis settings. Statistical analyses of read depths and balance ratios for the 165 AI-SNPs were calculated using the OriginPro 2021b software (version 9.8, OriginLab Corporation, Northampton, MA, USA).

Sanger Sequencing of Locus rs7722456
DNA samples with an 'A' allele at locus rs7722456 were further confirmed by Sanger sequencing. Forward primer (5′-GCTGACTCTAGCCCTTTGGG-3′) and reverse primer (5′-GGTGGGTCTTGTTTGGCATT-3′) were synthesized and then used to amplify the locus rs7722456. The PCR products were separated by the method of agarose gel electrophoresis, and then purified using the DiaSpin DNA Gel Extraction kit (Sangon Biotech, Guangzhou, China) following manufacturer's instructions. Sanger sequencing was conducted at rs7722456 locus by Sangon Biotech (Sangon Biotech, Guangzhou, China).

Reference Populations
In the present study, 164 AI-SNP data sets of 26 populations were acquired from the 1000 Genomes Project Phase III database (locus rs10954737 was excluded from the population genetic studies as its genotype data were not available in the 1000 Genomes Project) [27]. The 165 AIM-SNP data sets for 22 populations were obtained from HGDP-CEPH database [28]. SNP genotype data for nine populations (Sichuan Tibetan, Qinghai Tibetan and Liangshan Yi [29], Hainan Han, Hainan Li and Gelao [23], Wuzhong Hui [30], Chinese Kazak [31], and Basque [32]) were obtained from previously published literature. Detailed information, such as population names and their abbreviations, population sizes of all reference populations, is listed in Supplementary Table 1.

Population Genetic Analyses
The linkage disequilibrium (LD) test, allelic frequencies and forensic statistical parameters of the 165 AI-SNP loci in the Gannan Tibetan group were calculated using STRAF [33]. Tests of Hardy-Weinberg equilibrium (HWE) for 165 AI-SNP loci in Gannan Tibetan group were performed by the Arlequin software (version 3.5.1.2, Laurent Excoffier & Heidi Lischer, Berne, Switzerland) [34] . The heatmap for the minimal allelic frequencies (MAF) of 164 AI-SNP loci was created using the R software (version 4.0.5, R Foundation for Statistical Computing, Vienna, Austria). Pairwise fixation index (F ST ) values, which were used to measure the genetic distances of pairwise populations, were calculated between Gannan Tibetan group and reference populations using the Arlequin software (version 3.5.1.2, Excoffier & Lischer, Berne, Switzerland) [34]. Moreover, Nei's genetic distances (D A ) between the Gannan Tibetan group and other reference populations were calculated using the DISPAN program (Nei & Tajima & Tateno, Kanagawa, Japan) [35]. A neighbor-joining (NJ) phylogenetic tree was constructed on the basis of pairwise D A values by the MEGA software (version 7, Sudhir Kumar & Glen Stecher & Koichiro Tamura, PA, USA) [36], and then visualized with the ggtree package (version 3.4.2, Guangchuang Yu, Guangdong, China) [37] from the R software (version 4.0.5, R Foundation for Statistical Comput-ing, Vienna, Austria). Phylogenetic trees constructed using the maximum likelihood (ML) method were generated using the TreeMix software (version 1.13, Joseph K. Pickrell & Jonathan K. Pritchard, IL, USA), which assumed that 0-10 migration events happened among all the populations [38]. Principal component analyses (PCA) were also carried out to evaluate the genetic relationships between the Gannan Tibetan group and reference groups. PCAs at individual scale were performed based on the genotype data of 164 AI-SNP loci of Gannan Tibetan group and reference populations using the R software and the SmartPCA package implemented in the EIGENSOFT software (version 6.

Assessment for the NGS Results
In this study, all 165 AI-SNP genotypes in 101 Gannan Tibetan individuals and positive control DNA were successfully generated using the Ion S5™ XL system. Four Ion™ 530 chips generated a total of 1.5~1.87 gigabases (Gb). Ion sphere particle (ISP) loading rate of each Ion™ 530 chip was more than 78%. As recommended by the manufacturer's instructions, total reads should be greater than 1 Gb; the ISP loading rate should be greater than 50%; and the total usable reads should be greater than 30%. These sequencing metrics all met the recommended values of the manufacturer's instructions. Read depths of 165 AI-SNP loci in 101 Gannan Tibetan individuals are shown in Supplementary Fig. 1. The minimum depth of coverage was 24×, which was noted at rs9845457 locus in sample GNZ13, while the maximum read depth was 8287×, which was seen at rs7657799 locus in GNZ54. The mean read depths for all 101 individuals ranged from 231 ± 87× (mean ± standard deviation) to 2932 ± 1359×. The balance ratios of all amplicons from the forward direction in the 101 samples are shown in Fig. 1, and the box plots revealed that the balance ratios of almost all amplicons were around 0.5.
Sixty-three individuals (accounting for 62.4% of total tested Gannan Tibetan individuals) were genotyped 'AT' at rs7722456. Genotypes of rs7722456 at these samples were further confirmed by Sanger sequencing. Results of Sanger sequencing showed that the genotypes in these 63 individuals were 'TT' at rs7722456 (Supplementary Fig. 2).

HWE and LD Tests of 165 AI-SNPs in the Gannan Tibetan Group
Before the population genetic investigations, the HWE tests of the 165 AI-SNPs were carried out in the Gannan Tibetan group, and the results are shown in Supplementary Table 2. The rs1462906, rs1871534, rs2814778, rs3916235, rs4880436, rs4891825, rs6754311, rs7326934 and rs7722456 of 165 AI-SNPs were excluded from the HWE tests because of their homozygous genotypes. The results of HWE tests showed that no significant deviations from HWE for the remaining 156 AI-SNPs were observed in the Gannan Tibetan group after sequential Bonferroni corrections (p = 0.05/156 = 0.0003).
The LD tests for pairwise SNP loci were performed based on the genotype data, and the p-values are shown in Supplementary Table 3. Similarly, rs1462906, rs1871534, rs2814778, rs3916235, rs4880436, rs4891825, rs6754311, rs7326934 and rs7722456 were also excluded from the LD tests because of their homozygous genotypes. After sequential Bonferroni corrections (p = 0.05/24150 = 2.07 × 10 −6 ), all pairwise SNP loci reached to the state of linkage equilibrium in the studied Gannan Tibetan group except for the pair of rs1229984 and rs3811801 (p = 8.72 × 10 −19 ).

Analyses of Genetic Ancestry of the Gannan Tibetan Group on the Basis of 164 AI-SNP Loci
Admixture predictions and population likelihood analyses for 101 Gannan Tibetans were first conducted using the plugin in Torrent Suite, and all individuals were predicted as East Asian individuals. When trying to predict the ancestral information of East Asian subgroups using this panel, the prediction results are not particularly ac- curate (Supplementary Fig. 3). Genetic ancestry analyses for the Gannan Tibetan group were evaluated based on the 164 AI-SNP loci using both the ADMIXTURE (version 1.  Fig. 2A,B, respectively. The smallest CV error value was found when K = 5 (Fig. 2B, CV error = 0.50892). Therefore, we adopted the K = 5 as the optimal K value, and all individuals were assigned to five different clusters, which could differentiate among African (a cyanbased ancestry cluster), American (a mixture of purple and green), South Asian (a brick-red-based ancestry cluster), East Asian (an orange-based ancestry cluster) and European populations (a purple-based ancestry cluster), as shown in Fig. 2A.
The Bayesian structure analyses indicated that the Gannan Tibetan group shared similar genetic ancestry structure with East Asian populations when K values were set to 3~5. Cluster analysis also showed that the Gannan Tibetans clustered tightly with the East Asian individuals (Fig. 2C). At K = 6, subcluster with pink-based ancestry structure was identified in Tibeto-Burman groups such as the Gannan Tibetan group, the Sichuan Tibetan group and the Liangshan Yi group. The Gannan Tibetan group displayed a mixture of pink and orange ancestry structure, which were different from Han populations in flat land. The studied Gannan Tibetan group shared similar ancestry structure with groups residing in its adjacent areas, such as the Sichuan Tibetan group and the Liangshan Yi group.

Heatmap and PCA plots among the Gannan Tibetan Group and Reference Populations Based on Ancestral Informative SNP Markers
To visualize the distributions of allele frequencies of 164 AI-SNPs (locus rs10954737 was excluded due to its unavailability in the 1000 Genomes Project) among the Gannan Tibetan group and reference populations, a heatmap was constructed, and the results are shown in Fig. 3. In the heatmap, the gray to green color scheme represented low to high allele frequencies of the 164 AI-SNP loci. Worldwide reference populations could be distinguished to four major clusters: (1) twelve populations from Europe clustered together into subbranch I; (2) most populations from Southern Asia gathered together and formed subbranch II; (3) most reference populations from China clustered together into subbranch III while Chinese Kazak group clustered between European and East Asian populations. At the same time, the studied Gannan Tibetan group clustered closely with Chinese groups, especially with the Sichuan Tibetan group; (4) eleven populations from Africa gathered into the subbranch IV. Meanwhile, the 164 AI-SNP loci could be divided into six subclusters (clusters A to F). SNP loci in clusters A and E displayed low allelic frequencies in the African populations, and these loci could distinguish the African populations from other continental reference pop-ulations; cluster D contained a set of SNP loci with low allelic frequencies in the East Asian populations and relatively large allelic frequencies in other continental reference populations; SNP loci in the subbranch F-1 showed higher allelic frequencies in African populations compared with other reference populations, and these loci could also  Fig. 4A,B, individuals from worldwide populations gathered into four major clusters: Africa, Europe, East Asia and South Asia, labeled in red, blue, green and magenta, respectively. The studied Gannan Tibetan individuals clustered closely with the East Asian individuals, especially with the Chinese groups. A 3D-PCA plot was also generated using an online tool based on the PC1, PC2 and PC3, and the result is shown in Fig. 4C. The top three principal components could explain 44.19% total variance. Individuals from Africa, Europe, Oceania, South Asia, East Asia, Middle East and Americas were clustered together according to their geographical origins. Gannan Tibetan individuals clustered closely with East Asian individuals. A PCA plot at population scale was also generated (Fig. 4D). PC1 and PC2 could explain 77.2% total variance, and the studied Gannan Tibetan group was located closely with East Asian populations.

Phylogenetic Analyses and Genetic Differentiations between the Gannan Tibetan Group and the Reference Populations
Phylogenetic analyses were conducted using the NJ and ML methods to investigate the genetic relations between the Gannan Tibetan group and the reference populations, and the results are shown in Fig. 5. A NJ tree was constructed based on the pairwise D A values, and the length of the connecting line in the bar plot around the NJ tree represented the pairwise F ST values between Gannan Tibetan group and the reference populations (Fig. 5A). In the NJ tree, populations from Africa, Europe, South Asia and East Asia clustered together and formed four distinct subbranches accordingly. The studied Gannan Tibetan group was located in the East Asian subbranch, and clustered closely with the Sichuan Tibetan group and the Liangshan Yi group. In Fig. 5B, a rooted ML tree was constructed based on the genotypes of the 164 AI-SNPs. The obtained results were generally consistent with the NJ tree which demonstrated that the Gannan Tibetan group has tight phylogenetic connections with the East Asian populations, especially with the groups residing in its adjacent geographical areas.
We also calculated the pairwise F ST values between Gannan Tibetan group and the continental reference populations, and the results are shown in the circular barchart around the NJ tree. Large F ST values were observed between the Gannan Tibetan group and African populations. Moreover, the maximum F ST value was observed between the Gannan Tibetan group and Yoruba in Ibadan, Nigeria (F ST = 0.4664). Smaller F ST values were found between the Gannan Tibetan group and East Asian populations, and the minimum F ST value was found between the Gannan Tibetan group and the Sichuan Tibetan group.

Discussion
The read depths, strand balance, noise and other NGS quality control are important to evaluate the accuracy of NGS genotyping. Therefore, we first evaluated the sequencing metrics before the population genetic analyses. According to "Forensic sciences -Specifications for second generation sequencing-based DNA examination" (GA/T  1693-2020), sequencing depth of SNP loci should be larger than 100×. In this study, the minimum depth was higher than the analysis threshold recommended by the guideline, the average sequencing depth was greater than 200×; and the strand balances of most amplicons were about 50%, indicating that the NGS genotyping results were reliable. In addition, relative low depth and notable inter-locus imbalance were observed at a few of AI-SNP loci in the Gannan Tibetan group. According to the validation studies and the population genetic researches based on this panel, notable inter-locus imbalance was also observed in the published articles, which might be related to the flanking sequence or the primers of the SNPs [23,29,42,43].
When we uploaded the genotype data to an online tool-Snipper (http://mathgene.usc.es/snipper/), the website reminded us to check the "A" allele at rs7722456 locus in the present study because only C and T alleles were observed at rs7722456 locus in their database. And then we searched the population genotype data of this SNP locus on 1000 Genomes Project Phase III, and also found that only C and T alleles were observed at rs7722456 locus. In the present study, allele 'A' at rs7722456 (chr 5:170775980) was found in 63 Gannan Tibetan individuals, and reads for the 'A' allele accounted for approximate 20% of the total reads. After sequencing the chr5:170775853 -170776519 region, we found that a poly-A region [5 ′ -AATTACAGAT(C/T)AAAAAATACA-3 ′ ] was located immediately downstream of rs7722456 loci, which made it difficult for the Ion S5 XL system to identify the real number of poly-nucleotides in this region [42]. Certainly, raising the heterozygote balance threshold should be helpful to eliminate this deficiency [42,44].
Among the 165 AI-SNPs tested in this study, only one pair of rs1229984 and rs3811801 loci showed linkage disequilibrium, which was mainly caused by the inheritance of a haplotype encompassing these two SNP loci. Loci rs1229984 (chr 4:99318162, coding for the Arg48His substitution) and rs3811801 (chr 4:99323162) belong to the core haplotype of alcohol dehydrogenase family 1B gene (ADH1B), which was associated with the catalytic activity of the enzyme alcohol dehydrogenase [4]. Forensic parameters of the 165 AI-SNPs indicated that not all of the SNP loci showed high genetic polymorphisms in the Gannan Tibetan group. The cumulative random match probability of the 164 AI-SNPs was determined to be 7.269 × 10 −47 , which was greater than that of the 27 autosomal STRs in the ForenSeq DNA Signature Prep Kit [45,46]. The CPD and CPE values of the 164 AI-SNPs in Gannan Tibetan group indicated that this panel could be an effective tool in the forensic individual identification and parentage testing of the Gannan Tibetan group.
The origin, migration and genetic background of Tibetan group have become the research hotspots in the field of population genetics, molecular anthropology, archaeology and linguistics [8,[47][48][49]. In this study, we investigated the genetic ancestry structure of the Gannan Tibetan group on the basis of a set of AI-SNP loci. Results of Bayesian structure analyses indicated that the ancestral structure of the Gannan Tibetan group was dominated by the East Asian component, and the Gannan Tibetan group shared similar genetic ancestry structure with the Sichuan Tibetan group and the Liangshan Yi group.
To further analyze the genetic relationships among Gannan Tibetan group and reference populations, allelic frequency distributions, PCA, genetic distances and phylogenetic analyses were also conducted based on the shared AI-SNPs in the studied Gannan Tibetan group and continental reference populations. These analyses confirmed that the AI-SNP loci included in the Precision ID Ancestry Panel could be a valuable tool for the ancestral predictions of African, European, South Asian and East Asian populations. When trying to predict the ancestral information of East Asian subpopulations using this panel, the prediction results are not particularly accurate. An extended set of SNPs would be required to distinguish within East Asian subpopulations.
Results of genetic affinity analyses indicated that the Gannan Tibetan group had closer genetic affinities with East Asian populations, especially tighter genetic ties with those groups residing in its neighboring geographical regions like the Sichuan Tibetan group and the Liangshan Yi group. Many researchers estimated that the Sino-Tibetan groups shared a common ancestor who originated from the upper and middle reaches of Yellow River. With the spread of agriculture, ancient Han Chinese and Tibetan group derived from the shared ancestors about 5900 years ago [49][50][51]. Linguistic study and archaeological evidence also inferred that Tibeto-Burman populations consecutively migrated southward to be dispersed along the Tibetan-Yi ethnic corridor into the western Sichuan and western Yunnan provinces [49,52]. Population genetic evidences also demonstrated that the studied Gannan Tibetan group had closer genetic relationships with East Asian populations, which might be due to the extensive gene exchanges among Tibetan, Han Chinese and other groups residing around the Tibetan Plateau [2,29,47]. Besides, the plateau geographical environment also shaped the unique genetic characteristics of the Tibetan group. Frequencies of SNP haplotypes associated with high-altitude hypoxia adaptation were higher than those in populations residing in the plain region [53][54][55]. Therefore, these SNP loci mentioned above might be regarded as AIM-SNP markers to distinguish the Tibetan group and Han populations. Population genetic studies based on Y-SNP haplogroups showed that the Tibetan group was dominated by D-M174 haplogroup, followed by O-M175 haplogroup, which had relative high frequencies in Han population. The mtDNA haplogroups (M9a'b, G, D and F) of East Asian origin had high frequencies in the Tibetan group, which also indicated the close genetic relationships among the Tibetan group and East Asian popula-tions [56]. In the future, more types of genetic markers will be used for population genetic analysis, which will allow us to have a deeper understanding of the population genetic sub-structures of the Tibetan groups in Chinese different regions.

Conclusions
In this study, the NGS performance and forensic statistical parameters of the 165 AI-SNP loci in the Gannan Tibetan group were evaluated. The 165 AI-SNP loci showed the varying degrees of genetic polymorphisms in the Gannan Tibetan group, and the combined use of these loci could be an effective tool in the forensic individual identification and parentage testing of the Gannan Tibetan group. The ancestral information component of the Gannan Tibetan group was dominated by the East Asian component. Population genetic analyses indicated that the Gannan Tibetan group had close genetic affinities with East Asian populations compared with other intercontinental reference populations, especially tighter genetic relationships with the groups residing in its neighboring geographical regions.

Availability of Data and Materials
The raw genotype data used and analyzed during the current study are available from the corresponding author on reasonable request.

Author Contributions
BZ designed this research and were responsible for all the processes of this research. WC and MC conducted the experiment and analyzed the raw data. WC wrote this manuscript. HY collected the samples. QY and LC provided technical support for the experiments. LC, LL and XB assisted the experiment. BZ, MC, LL, LC and XB revised this manuscript. All authors have read and agreed to submit the manuscript.

Ethics Approval and Consent to Participate
This study has been approved by the ethics committee of Xi'an Jiaotong University Health Science Center and Southern Medical University (Ethical Approval Number: 2019-1039). This research was conducted in accordance with the ethical principle for medical research involving human subjects recommended by the World Medical Association Declaration of Helsinki. Sample collections and sequencing experiments presented herein were performed in strict compliance with the ethical regulation. Every volunteer signed a written informed consent before sample collection.