Nanopore sequencing of forensic short tandem repeats using QNome of Qitan Technology.

Devices of nanopore sequencing can be highly portable and of low cost. Thus, nanopore sequencing is promising in in-field forensic applications. Previous investigations have demonstrated that nanopore sequencing is feasible for genotyping forensic short tandem repeats (STRs) by using sequencers of Oxford Nanopore Technologies. Recently, Qitan Technology launched a new portable nanopore sequencer and became the second supplier in the world. Here, for the first time, we assess the QNome (QNome-3841) for its accuracy in nanopore sequencing of STRs and compare with MinION (MinION Mk1B). We profile 54 STRs of 21 unrelated individuals and 2800M standard DNA. The overall accuracy for diploid STRs and haploid STRs were 53.5% (378 of 706) and 82.7% (134 of 162), respectively, by using QNome. The accuracies were remarkably lower than those of MinION (diploid STRs, 84.5%; haploid, 90.7%), with a similar amount of sequencing data and identical bioinformatics analysis. Although it was not reliable for diploid STRs typing by using QNome, the haploid STRs were consistently correctly typed. The majority of errors (58.8%) in QNome-based STR typing were one-repeat deviations of repeat units in the error from true allele, related with homopolymers in repeats of STRs.

Multiple Human Population Movements and Cultural Dispersal Events Shaped the Landscape of Chinese Paternal Heritage.

Large-scale genomic projects and ancient DNA innovations have ushered in a new paradigm for exploring human evolutionary history. However, the genetic legacy of spatiotemporally diverse ancient Eurasians within Chinese paternal lineages remains unresolved. Here, we report an integrated Y-chromosome genomic database encompassing 15,563 individuals from both modern and ancient Eurasians, including 919 newly reported individuals, to investigate the Chinese paternal genomic diversity. The high-resolution, time-stamped phylogeny reveals multiple diversification events and extensive expansions in the early and middle Neolithic. We identify four major ancient population movements, each associated with technological innovations that have shaped the Chinese paternal landscape. First, the expansion of early East Asians and millet farmers from the Yellow River Basin predominantly carrying O2/D subclades significantly influenced the formation of the Sino-Tibetan people and facilitated the permanent settlement of the Tibetan Plateau. Second, the dispersal of rice farmers from the Yangtze River Valley carrying O1 and certain O2 sublineages reshapes the genetic makeup of southern Han Chinese, as well as the Tai-Kadai, Austronesian, Hmong-Mien, and Austroasiatic people. Third, the Neolithic Siberian Q/C paternal lineages originated and proliferated among hunter-gatherers on the Mongolian Plateau and the Amur River Basin, leaving a significant imprint on the gene pools of northern China. Fourth, the J/G/R paternal lineages derived from western Eurasia, which were initially spread by Yamnaya-related steppe pastoralists, maintain their presence primarily in northwestern China. Overall, our research provides comprehensive genetic evidence elucidating the significant impact of interactions with culturally distinct ancient Eurasians on the patterns of paternal diversity in modern Chinese populations.

Circadian metabolites for evaluating the timing of bloodstain deposition: A preliminary study.

Metabolites, as products of cellular metabolism, can provide a wealth of biological information and are less susceptible to degradation than other biomarkers due to their low molecular weight. Due to these properties, metabolites can be used as valuable biomarkers for forensic investigations. Knowing the timing of deposition of bloodstain could help to reconstruct crime scenes, draw conclusions about the time of the crime, and narrow down the circle of possible suspects. Previous studies have indicated that the concentration of some metabolites in blood is subject to circadian changes. However, the circadian metabolites of bloodstains have been still unclear. A total of sixty-four bloodstain samples were prepared under real conditions in three time categories (morning/noon (09:00 h ∼ 17:00 h), afternoon/evening (18:00 h ∼ 23:00 h) and night/early morning (24:00 h ∼ 08:00 h)). Fifty metabolites of bloodstains with significant differences were identified in the three time categories. Twenty-eight of these metabolites exhibited significant circadian changes. Finally, three independently contributing circadian metabolites were selected to build the logistic regression model, with an area under the curve of 0.91, 0.84 and 0.87 for the prediction of bloodstain deposition time in the morning/noon, afternoon/evening and night/early morning, respectively. The study indicated that circadian metabolites can be used for evaluating the timing of bloodstain deposition. This would provide a valuable perspective for analyzing the deposition time of biological traces in forensic investigations.

Case reports on uniparental disomy of chromosomes 6 and 3 in paternity testing.

In paternity testing, when there are Mendelian errors in the alleles between the child and the parents, a slippage mutation, or silent allele may not fully explain the phenomenon. Sometimes, it is attributed to chromosomal abnormalities, such as uniparental disomy (UPD). Here, we present the investigation of two cases of suspected UPD in paternity testing based on short tandem repeat (STR) detection (capillary electrophoresis platform). Case 1 involves a trio, where all genotypes detected on chromosome 6 in the child are homozygous and found in the father. Case 2 is a duo (mother and child), where all genotypes on chromosome 3 in the child are homozygous and not always found in the mother. At the same time, Mendelian error alleles were also observed at specific loci in these two chromosomes. Furthermore, we used the MGIEasy Signature Identification Library Prep Kit for sequencing on the massively parallel sequencing platform, which included common autosomal, X and Y chromosomes, and mitochondrial genetic markers used in forensic practice. The results showed that the genotypes of shared STRs on the two platforms were consistent, and STRs and single nucleotide polymorphisms (SNPs) on these two chromosomes were homozygous. All other genetic markers followed the laws of inheritance. A comprehensive analysis supported the parent-child relationship between the child and the alleged parent, and the observed genetic anomalies can be attributed to UPD. UPD occurrences are rare, and ignoring its presence can lead to erroneous exclusions in paternity testing, particularly when multiple loci on a chromosome exhibit homozygosity.

Male-specific age prediction based on Y-chromosome DNA methylation with blood using pyrosequencing.

Age prediction is an important aspect of forensic science that offers valuable insight into identification. In recent years, extensive studies have been conducted on age prediction based on DNA methylation, and numerous studies have demonstrated that DNA methylation is a reliable biomarker for age prediction. However, almost all studies on age prediction based on DNA methylation have focused on age-related CpG sites in autosomes, which are concentrated on single-source DNA samples. Mixed samples, especially male-female mixed samples, are common in forensic casework. The application of Y-STRs and Y-SNPs can provide clues for the genetic typing of male individuals in male-female mixtures, but they cannot provide the age information of male individuals. Studies on Y-chromosome DNA methylation can address this issue. In this study, we identified five age-related CpG sites on the Y chromosome (Y-CpGs) and developed a male-specific age prediction model using pyrosequencing combined with a support vector machine algorithm. The mean absolute deviation of the model was 5.50 years in the training set and 6.74 years in the testing set. When we used a male blood sample to predict age, the deviation between the predicted and chronological age was 1.18 years. Then, we mixed the genomic DNA of the male and a female at ratios of 1:1, 1:5, 1:10, and 1:50, the range of deviation between the predicted and chronological age of the male in the mixture was 1.16-1.74 years. In addition, there was no significant difference between the methylation values of bloodstains and blood in the same sample, which indicates that our model is also suitable for bloodstain samples. Overall, our results show that age prediction using DNA methylation of the Y chromosome has potential applications in forensic science and can be of great help in predicting the age of males in male-female mixtures. Furthermore, this work lays the foundation for future research on age-related applications of Y-CpGs.

Malaria resistance-related biological adaptation and complex evolutionary footprints inferred from one integrative Tai-Kadai-related genomic resource.

Pathogen‒host adaptative interactions and complex population demographical processes, including admixture, drift, and Darwen selection, have considerably shaped the Neolithic-to-Modern Western Eurasian population structure and genetic susceptibility to modern human diseases. However, the genetic footprints of evolutionary events in East Asia remain unknown due to the underrepresentation of genomic diversity and the design of large-scale population studies. We reported one aggregated database of genome-wide SNP variations from 796 Tai-Kadai (TK) genomes, including that of Bouyei first reported here, to explore the genetic history, population structure, and biological adaptative features of TK people from southern China and Southeast Asia. We found geography-related population substructure among TK people using the state-of-the-art population genetic structure reconstruction techniques based on the allele frequency spectrum and haplotype-resolved phased fragments. We found that the northern TK people from Guizhou harbored one TK-dominant ancestry maximized in the Bouyei people, and the southern TK people from Thailand were more influenced by Southeast Asians and indigenous people. We reconstructed fitted admixture models and demographic graphs, which showed that TK people received gene flow from ancient southern rice farmer-related lineages related to the Hmong-Mien and Austroasiatic people and from northern millet farmers associated with the Sino-Tibetan people. Biological adaptation focused on our identified unique TK lineages related to Bouyei, which showed many adaptive signatures conferring Malaria resistance and low-rate lipid metabolism. Further gene enrichment, the allele frequency distribution of derived alleles, and their correlation with the incidence of Malaria further confirmed that CR1 played an essential role in the resistance of Malaria in the ancient "Baiyue" tribes.

Evaluation of reverse transcription yield of RNA standards and forensic samples based on droplet digital PCR.

RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 µg/µL - 0.24 ng/µL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 µL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments.

Visual identification for species and sex derived from bloodstain based on phosphate-mediated isothermal amplification colorimetric system.

Species and sex confirmation of the biological specimen play a crucial role in crime investigation. However, the specimen found in the scene is always trace quantity, which is hard to be analyzed by current methods. Moreover, the time-consuming DNA extraction, sophisticated apparatus, and complex data processing make it difficult to satisfy the demand of speediness and convenience for point-of-care tests. In this study, we first exhibit a phosphate-based visual system for field-based species and sex identification derived from trace bloodstain. By introducing phosphate ion-based colorimetry into loop-mediated isothermal amplification (LAMP) for result interpretation, not only the bloodstain can be directly submitted to mitochondrial variant amplification owing to the enhanced amplification efficiency by pyrophosphate ion hydrolyzation, but also the colorimetric signal can be recognized by the naked eye for result output within 30 min through molybdophosphate generation. Aerosol contamination, the major conflict of LAMP, has been solved once and for all by integrating uracil-DNA glycosylase into this system that still holds on a constant temperature. As a demonstration, cytochrome b and Y-chromosomal amelogenin are employed to identify species and sex respectively, which has achieved a highly sensitive and specific distinguishability under a strong interferential background. Accurate results can be obtained from both the simulative degraded and dated specimen, which indicates that this novel system may serve as a promising tool in forensic practice.

Nuclear RNA homeostasis promotes systems-level coordination of cell fate and senescence.

Understanding cellular coordination remains a challenge despite knowledge of individual pathways. The RNA exosome, targeting a wide range of RNA substrates, is often downregulated in cellular senescence. Utilizing an auxin-inducible system, we observed that RNA exosome depletion in embryonic stem cells significantly affects the transcriptome and proteome, causing pluripotency loss and pre-senescence onset. Mechanistically, exosome depletion triggers acute nuclear RNA aggregation, disrupting nuclear RNA-protein equilibrium. This disturbance limits nuclear protein availability and hinders polymerase initiation and engagement, reducing gene transcription. Concurrently, it promptly disrupts nucleolar transcription, ribosomal processes, and nuclear exporting, resulting in a translational shutdown. Prolonged exosome depletion induces nuclear structural changes resembling senescent cells, including aberrant chromatin compaction, chromocenter disassembly, and intensified heterochromatic foci. These effects suggest that the dynamic turnover of nuclear RNA orchestrates crosstalk between essential processes to optimize cellular function. Disruptions in nuclear RNA homeostasis result in systemic functional decline, altering the cell state and promoting senescence.

Differentiated adaptative genetic architecture and language-related demographical history in South China inferred from 619 genomes from 56 populations.

BACKGROUND: The underrepresentation of human genomic resources from Southern Chinese populations limited their health equality in the precision medicine era and complete understanding of their genetic formation, admixture, and adaptive features. Besides, linguistical and genetic evidence supported the controversial hypothesis of their origin processes. One hotspot case was from the Chinese Guangxi Pinghua Han people (GPH), whose language was significantly similar to Southern Chinese dialects but whose uniparental gene pool was phylogenetically associated with the indigenous Tai-Kadai (TK) people. Here, we analyzed genome-wide SNP data in 619 people from four language families and 56 geographically different populations, in which 261 people from 21 geographically distinct populations were first reported here. RESULTS: We identified significant population stratification among ethnolinguistically diverse Guangxi populations, suggesting their differentiated genetic origin and admixture processes. GPH shared more alleles related to Zhuang than Southern Han Chinese but received more northern ancestry relative to Zhuang. Admixture models and estimates of genetic distances showed that GPH had a close genetic relationship with geographically close TK compared to Northern Han Chinese, supporting their admixture origin hypothesis. Further admixture time and demographic history reconstruction supported GPH was formed via admixture between Northern Han Chinese and Southern TK people. We identified robust signatures associated with lipid metabolisms, such as fatty acid desaturases (FADS) and medically relevant loci associated with Mendelian disorder (GJB2) and complex diseases. We also explored the shared and unique selection signatures of ethnically different but linguistically related Guangxi lineages and found some shared signals related to immune and malaria resistance. CONCLUSIONS: Our genetic analysis illuminated the language-related fine-scale genetic structure and provided robust genetic evidence to support the admixture hypothesis that can explain the pattern of observed genetic diversity and formation of GPH. This work presented one comprehensive analysis focused on the population history and demographical adaptative process, which provided genetic evidence for personal health management and disease risk prediction models from Guangxi people. Further large-scale whole-genome sequencing projects would provide the entire landscape of southern Chinese genomic diversity and their contributions to human health and disease traits.

Body fluids should be identified before estimating the time since deposition (TsD) in microbiome-based stain analyses for forensics.

Identification and the time since deposition (TsD) estimation of body fluid stains from a crime scene could provide valuable information for solving the cases and are always difficult for forensics. Microbial characteristics were considered as a promising biomarker to address the issues. However, changes in the microbiota may damage the specific characteristics of body fluids. Correspondingly, incorrect body fluid identification may result in inaccurate TsD estimation. The mutual influence is not well understood and limited the codetection. In the current study, saliva, semen, vaginal secretion, and menstrual blood samples were exposed to indoor conditions and collected at eight time points (from fresh to 30 days). High-throughput sequencing based on the 16S rRNA gene was performed to characterize the microbial communities. The results showed that a longer TsD could decrease the discrimination of different body fluid stains. However, the accuracies of identification still reached a quite high value even without knowing the TsD. Correspondingly, the mean absolute error (MAE) of TsD estimation significantly increased without distinguishing the types of body fluids. The predictive TsD of menstrual blood reached a quite low MAE (1.54 ± 0.39 d). In comparison, those of saliva (6.57 ± 1.17 d), semen (6.48 ± 1.33 d), and vaginal secretion (5.35 ± 1.11 d) needed to be further improved. The great effect of individual differences on these stains limited the TsD estimation accuracy. Overall, microbial characteristics allow for codetection of body fluid identification and TsD estimation, and body fluids should be identified before estimating TsD in microbiome-based stain analyses.IMPORTANCEEmerged evidences suggest microbial characteristics could be considered a promising tool for identification and time since deposition (TsD) estimation of body fluid stains. However, the two issues should be studied together due to a potential mutual influence. The current study provides the first evidence to understand the mutual influence and determines an optimal process for codetection of identification and TsD estimation for unknown stains for forensics. In addition, we involved aged stains into our study for identification of body fluid stains, rather than only using fresh stains like previous studies. This increased the predictive accuracy. We have preliminary verified that individual differences in microbiotas limited the predictive accuracy of TsD estimation for saliva, semen, and vaginal secretion. Microbial characteristics could provide an accurate TsD estimation for menstrual blood. Our study benefits the comprehensive understanding of microbiome-based stain analyses as an essential addition to previous studies.

A Bibliometric and Visual Analysis of the Current Status and Trends of Forensic Mixed Stain Research.

OBJECTIVES: To explore the context and hotspot changes of forensic mixed stain research through bibliometric approach. METHODS: The literature of forensic mixed stain included in the core collection of Web of Science database from 2011 to 2022 were collected as the study object, and the annual publication number, countrie (region), institution, journal, keywords, etc. were bibliometrically and visually analyzed using the R-based Bibliometrix 1.1.6 package and VOSviewer 1.6.18 software. RESULTS: A total of 732 articles on forensic mixed stain were included from 2011 to 2022, with the annual number of articles published and the annual citation frequency showing a steady increase year by year. Among the 59 countries (regions) with the most published articles, the United States ranked first with 246 articles, followed by China with 153 articles. The literature came from 104 journals, and the total number of articles published in the top 10 journals was 633. FORENSIC SCI INT GENET ranked first with 307 articles. Visual analysis using VOSviewer software showed that keywords could be divided into four research clusters, namely the genetic marker development group (blue), the mixed stain typing analysis theory group (red), the sequencing analysis group (yellow), and the case sample research group (green). It can be divided into four development stages in terms of different time periods: early development (2011-2013), middle development (2014-2016), rapid development (2017-2020) and latest development (2021-2022). CONCLUSIONS: The number of publications by domestic and foreign scholars in the study of mixed stain in forensic science is showing a relatively stable trend. Machine learning, next generation sequencing and other research have been the hottest topics that have attracted the most attention in recent years, which is expected to further develop the theory of mixed stain typing and sequencing analysis in forensic mixed stain research.

The Latest Research Progress on Cell-Free DNA and Prospects of Its Forensic Application.

In recent years, with the continuous progress of DNA extraction and detection technology, cell-free DNA(cfDNA)has been widely used in the life science field, and its potential application value in forensic identification is becoming more and more obvious. This paper reviews the concept, formation mechanism, and classification of cfDNA, etc., and describes the latest research progress of cfDNA in personal identification of crime scene touch DNA samples and non-invasive prenatal paternity testing (NIPPT). Meanwhile, this paper summarizes the potential application of cfDNA in injury inference, and discusses the advantages and disadvantages of common cfDNA analysis methods and techniques, and its application prospects, to provide a new idea for the wide application of cfDNA in the field of forensic science.

Pilot validation of on-field STR typing and human identity testing by MinION nanopore sequencing.

Nanopore sequencing technology has broad application prospects in forensic medicine due to its small size, portability, fast speed, real-time result analysis capabilities, single-molecule sequencing abilities, and simple operation. Here, we demonstrate for the first time that nanopore sequencing platforms can be used to identify individuals in the field. Through scientific and reasonable design, a nanopore MinION MK1B device and other auxiliary devices are integrated into a portable detection box conducive to individual identification at the accident site. Individual identification of 12 samples could be completed within approximately 24 h by jointly detecting 23 short tandem repeat (STR) loci. Through double-blinded experiments, the genotypes of 49 samples were successfully determined, and the accuracy of the STR genotyping was verified by the gold standard. Specifically, the typing success rate for 1150 genotypes was 95.3%, and the accuracy rate was 86.87%. Although this study focused primarily on demonstrating the feasibility of full-process testing, it can be optimistically predicted that further improvements in bioinformatics workflows and nanopore sequencing technology will help enhance the feasibility of Oxford Nanopore Technologies equipment for real-time individual identification at accident sites.

Roles of Myokines and Muscle-Derived Extracellular Vesicles in Musculoskeletal Deterioration under Disuse Conditions.

Prolonged inactivity and disuse conditions, such as those experienced during spaceflight and prolonged bedrest, are frequently accompanied by detrimental effects on the motor system, including skeletal muscle atrophy and bone loss, which greatly increase the risk of osteoporosis and fractures. Moreover, the decrease in glucose and lipid utilization in skeletal muscles, a consequence of muscle atrophy, also contributes to the development of metabolic syndrome. Clarifying the mechanisms involved in disuse-induced musculoskeletal deterioration is important, providing therapeutic targets and a scientific foundation for the treatment of musculoskeletal disorders under disuse conditions. Skeletal muscle, as a powerful endocrine organ, participates in the regulation of physiological and biochemical functions of local or distal tissues and organs, including itself, in endocrine, autocrine, or paracrine manners. As a motor organ adjacent to muscle, bone tissue exhibits a relative lag in degenerative changes compared to skeletal muscle under disuse conditions. Based on this phenomenon, roles and mechanisms involved in the communication between skeletal muscle and bone, especially from muscle to bone, under disuse conditions have attracted widespread attention. In this review, we summarize the roles and regulatory mechanisms of muscle-derived myokines and extracellular vesicles (EVs) in the occurrence of muscle atrophy and bone loss under disuse conditions, as well as discuss future perspectives based on existing research.

Aptamer-based fluorescent sensor for highly sensitive detection of methamphetamine.

The construction of a fluorescence aptamer sensor was achieved by employing the fundamental principle of fluorescence resonance energy transfer. By employing molecular modeling technologies to identify the binding site, the high-affinity aptamer APT-40nt was derived from the whole sequence and utilized on the graphene oxide (GO) fluorescent platform for the purpose of achieving a highly sensitive detection of methamphetamine (METH). The aptamer tagged with fluorescein (FAM) dye undergoes quenching in the presence of GO due to π-stacking interaction. With the addition of the target, the aptamer that has been tagged was detached from the GO surface, forming a stable complex with METH. This process resulted in fluorescence restoration of the system, and the degree of fluorescence restoration was proportional to METH concentration in the linear range of 1-50 and 50-200 nM. Notably, under optimized conditions, the detection limit of this aptasensor was as low as 0.78 nM, which meets the detection limit requirements of METH detection in saliva and urine in some countries and regions. Moreover, other common illicit drugs and metabolites had minimizing interference with the determination. The established aptasensor, therefore, has been successfully applied to detect METH in saliva and urine samples and exhibited satisfactory recoveries (87%-111%). This aptasensor has the advantages of low detection limit, excellent selectivity, ease of operation, and low cost, providing a promising strategy for on-site detection of METH in saliva and urine.

DNA methylation profiling reveals potential biomarkers of ß-lactams induced fatal anaphylactic shock.

Anaphylaxis is a serious reaction of systemic hypersensitivity with that rapid onset and sudden death. Drug hypersensitivity, particularly induced by ß-lactams, is one of the most frequent causes of anaphylaxis in adults. But identification of anaphylactic shock, in forensic sciences recently, is difficult, because it mainly depends on nonspecific characteristic morphological changes, as well as exclusion and circumstantial evidence. Here, we detected DNA methylation signatures of ß-lactams-induced fatal anaphylactic shock with the Illumina Infinium Human Methylation EPIC BeadChip, to screen potential forensic biomarkers and reveal the molecular mechanisms of drug-induced anaphylaxis with fatal shock and sudden death. Our results indicated that DNA methylation was associated with ß-lactams-induced fatal anaphylactic shock, in which the hypomethylation played a vital role. We found that 1459 differentially methylated positions (DMPs) were mainly involved in ß-lactams-induced fatal anaphylactic shock by regulating MAPK and other signaling pathways. 18 DNA methylation signatures that could separate ß-lactams-induced anaphylactic shock from healthy individuals were identified. The altered methylation of DMPs can affect the transcription of corresponding genes and promote ß-lactams-induced fatal anaphylactic shock. The results suggest that DNA methylation can detect forensic identification markers of drug-induced anaphylaxis with fatal shock and sudden death, and it is an effective method for the forensic diagnosis.

Differentiated genomic footprints suggest isolation and long-distance migration of Hmong-Mien populations.

BACKGROUND: The underrepresentation of Hmong-Mien (HM) people in Asian genomic studies has hindered our comprehensive understanding of the full landscape of their evolutionary history and complex trait architecture. South China is a multi-ethnic region and indigenously settled by ethnolinguistically diverse HM, Austroasiatic (AA), Tai-Kadai (TK), Austronesian (AN), and Sino-Tibetan (ST) people, which is regarded as East Asia's initial cradle of biodiversity. However, previous fragmented genetic studies have only presented a fraction of the landscape of genetic diversity in this region, especially the lack of haplotype-based genomic resources. The deep characterization of demographic history and natural-selection-relevant genetic architecture of HM people was necessary. RESULTS: We reported one HM-specific genomic resource and comprehensively explored the fine-scale genetic structure and adaptative features inferred from the genome-wide SNP data of 440 HM individuals from 33 ethnolinguistic populations, including previously unreported She. We identified solid genetic differentiation between HM people and Han Chinese at 7.64‒15.86 years ago (kya) and split events between southern Chinese inland (Miao/Yao) and coastal (She) HM people in the middle Bronze Age period and the latter obtained more gene flow from Ancient Northern East Asians. Multiple admixture models further confirmed that extensive gene flow from surrounding ST, TK, and AN people entangled in forming the gene pool of Chinese coastal HM people. Genetic findings of isolated shared unique ancestral components based on the sharing alleles and haplotypes deconstructed that HM people from the Yungui Plateau carried the breadth of previously unknown genomic diversity. We identified a direct and recent genetic connection between Chinese inland and Southeast Asian HM people as they shared the most extended identity-by-descent fragments, supporting the long-distance migration hypothesis. Uniparental phylogenetic topology and network-based phylogenetic relationship reconstruction found ancient uniparental founding lineages in southwestern HM people. Finally, the population-specific biological adaptation study identified the shared and differentiated natural selection signatures among inland and coastal HM people associated with physical features and immune functions. The allele frequency spectrum of cancer susceptibility alleles and pharmacogenomic genes showed significant differences between HM and northern Chinese people. CONCLUSIONS: Our extensive genetic evidence combined with the historical documents supported the view that ancient HM people originated from the Yungui regions associated with ancient "Three-Miao tribes" descended from the ancient Daxi-Qujialing-Shijiahe people. Then, some have recently migrated rapidly to Southeast Asia, and some have migrated eastward and mixed respectively with Southeast Asian indigenes, Liangzhu-related coastal ancient populations, and incoming southward ST people. Generally, complex population migration, admixture, and adaptation history contributed to the complicated patterns of population structure of geographically diverse HM people.

Development of a novel panel for blood identification based on blood-specific CpG-linked SNP markers.

Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.