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Objectives: To establish the identification method of tumor tissue origin based on commonly used STR typing kits., Methods: ForenSeq TM DNA Signature Prep kit was used to detect the typing of 27 autosomal STR loci in 55 paired tumor tissue samples (tumor tissue paired with normal tissue of the same individual) and 75 unrelated individual whole blood samples. The genotyping data of full sibling pairs and parent-child pairs of 55 tumor tissues were simulated. The number of total identical alleles ( A n ) and identity by state (IBS) scores were calculated within the paired carcinoma (PC), the tumor-unrelated individual (UI), the tumor-simulated full sibling (FS) and the tumor-simulated parent-offspring (PO) groups. The tumor tissue origin identification and prediction models of 8 commonly used STR typing kits were established based on the statistical results obtained above, and an attempt was made to establish a specific model for tumor tissue origin identification. The accuracy, sensitivity and specificity were verified and evaluated using the additional 23 paired tumor tissue samples., Results: (1) In any kit, there was no statistically significant difference in the number of loci shared with 0 identical allele ( A 0 ) between the PC and PO groups. The number of loci shared with 1 identical allele ( A 1 ), 2 identical alleles ( A 2 ), and IBS scores were statistically significant different between the PC group and the UI, FS and PO groups. (2) The A n and IBS scores of different STRs varied in different groups. The A 2 levels of 13 STRs ( CSF1PO , D12S391 , D19S433 , D20S482 , D2S1338 , D3S1358 , D4S2408 , D7S820 , D8S1179 , FGA , TH01 , TPOX , vWA ) in PC group were higher than those of other STR loci. The A 2 levels of two STRs ( D6S1043 , Penta E ) in UI group were significantly lower than those of other STR loci. (3) The tumor tissue origin identification and prediction models of 8 commonly used STR typing kits and the identification model of tumor tissue origin with 15 STR loci (15-STRs) were successfully established, with sensitivity of 100%, specificity of 97.56%-99.88%, and accuracy of 97.59%-99.89%. Among them, the 15-STRs model had 100% sensitivity, 99.88% specificity, and 99.89% accuracy, which were higher than those of commonly used commercial kits., Conclusions: This study successfully establishes the tumor tissue origin identification methods with 8 commonly used STR typing kits, which expands the application of tumor tissue origin identification. In addition, the differences of different loci in the identification of tumor tissue origin were compared, and 15 STR loci which were particularly suitable for the identification of tumor tissue origin were selected, providing the data basis for the establishment of tumor origin tracing kits in future.

Objectives: To establish a rapid, accurate, and sensitive multiplex PCR detection method for the simultaneous identification of the six common edible meats (beef, lamp, chicken, pork, goose, duck), and to evaluate its application value in meat adulteration identification., Methods: Based on complete mitochondrial genomic sequences of six species in the GenBank database, DNA sequences (cattle： 16S rRNA ; sheep： COX-1 ; chickens： Cytb ; pig： COX-1 ; goose： NADH2 ; duck： 16S rRNA ) with intra-species conservation and inter-species specificity were screened, and species-specific primers were designed to construct a multiplex PCR detection system that can simultaneously detect the meat of six common species. The species specificity, sensitivity and reproducibility of the system were studied, and the simulated mixture sample detection was performed., Results: This study successfully constructed a multiplex PCR detection system that can detect the meats of six common species simultaneously. The system was not effective in DNA amplification of non-target species. When the DNA template sizes were 0.062 5-2 ng/μL, the amplified products of all six species could be detected. The duck component was still detected when the mixing ratio of duck and beef was as low as 0.5%., Conclusions: This study constructs and establishes a multiplex PCR detection system with strong specificity, high sensitivity, and good reproducibility. It can accurately identify the components of animal origin in common edible meats and provide a simple and practical method for identifying adulteration of common edible meats and meat products in China.

In the study of age estimation in living individuals, a lot of data needs to be analyzed by mathematical statistics, and reasonable medical statistical methods play an important role in data design and analysis. The selection of accurate and appropriate statistical methods is one of the key factors affecting the quality of research results. This paper reviews the principles and applicable principles of the commonly used medical statistical methods such as descriptive statistics, difference analysis, consistency test and multivariate statistical analysis, as well as machine learning methods such as shallow learning and deep learning in the age estimation research of living individuals, and summarizes the relevance and application prospects between medical statistical methods and machine learning methods. This paper aims to provide technical guidance for the age estimation research of living individuals to obtain more scientific and accurate results.

Objectives: To establish and forensically verify a 42 microhaplotypes (mircohaps, MHs) multiplex assay system based on next-generation sequencing (NGS), and to explore the application value of this system in the practice of forensic genetics., Methods: A total of 42 highly polymorphic MHs were selected from previous studies, and sequenced by the MiSeq FGx TM platform to verify the repeata-bility, sensitivity, specificity, stability, and mixture analysis ability of the detection system. Through population genetic investigation of 102 unrelated Chinese Han individuals in Liyang City, Jiangsu Province, China, the application value of this system in forensic genetics was evaluated., Results: The sequencing repeatability of the 42-plex MHs assay was 100% and the sensitivity was as low as 0.062 5 ng. The system had the ability to withstand the interference of indigo (≤2 500 ng/μL), humic acid (≤9 ng/μL), hemoglobin(≤20 μmol), and urea (≤200 ng/μL) and to detect mixtures of 2 people (1∶19), 3 people (1∶1∶9) and 4 people (1∶1∶1∶9). Based on 102 individual data, the combined power of discrimination and the combined power of exclusion were 1-3.45×10 -30 and 1-3.77×10 -11 , respectively, and the average effect value of alleles was 2.899., Conclusions: The 42-plex MHs assay was successfully established in this study and this system has high repeatability and sensitivity, good anti-jamming ability and mixture analysis ability. The 42 MHs are highly polymorphism and have good application value in individual identification and paternity testing.

Objectives: To explore the possibility of using human skin and oral microorganisms to estimate the geographic origin of an individual through the sequencing analysis of bacterial 16S rRNA gene., Methods: Microbial DNA was extracted from the palm and oral microorganisms of the Han population in Shanghai and Chifeng, Inner Mongolia, and the composition and diversity of the microbiota were analyzed by full-length 16S rRNA gene sequencing. Then, differential species were screened and a geographic location prediction model was constructed., Results: The compositions of palm and oral microorganisms between Shanghai and Chifeng samples were both different. The abundance and uniformity of palm side skin microorganisms were higher in Chifeng samples than in Shanghai samples, while there was no significant difference in oral microorganisms. Permutational multivariate analysis of variance (PERMANOVA) confirmed that th e β - diversity between the samples from the two places were statistically significant, and the coefficients of determination ( R 2 ) for skin and oral samples were 0.129 and 0.102, respectively. Through principal co-ordinates analysis (PCoA), the samples from the two places could be preliminarily distinguished. The predictive model had the accuracies of 0.90 and 0.83 for the geographic origin using the skin and oral samples, respectively., Conclusions: There are differences in the compositions of palm and oral microbiota between Han populations in Shanghai and Chifeng. The prediction model constructed by the random forest algorithm can trace the unknown individuals from the above two places.

Objectives: To explore the feasibility of genetic marker detection of semen-specific coding region single nucleotide polymorphism (cSNP) based on SNaPshot technology in semen stains and mixed body fluid identification., Methods: Genomic DNA (gDNA) and total RNA were extracted from 16 semen stains and 11 mixtures composed of semen and venous blood, and the total RNA was reverse transcribed into complementary DNA (cDNA). The cSNP genetic markers were screened on the validated semen-specific mRNA coding genes. The cSNP multiplex detection system based on SNaPshot technology was established, and samples were genotyped by capillary electrophoresis (CE)., Results: A multiplex detection system containing 5 semen-specific cSNPs was successfully established. In 16 semen samples, except the cSNP located in the TGM4 gene showed allele loss in cDNA detection results, the gDNA and cDNA typing results of other cSNPs were highly consistent. When detecting semen-venous blood mixtures, the results of cSNP typing detected were consistent with the genotype of semen donor and were not interfered by the genotype of venous blood donor., Conclusions: The method of semen-specific cSNPs detection by SNaPshot technology method can be applied to the genotyping of semen (stains) and provide information for determining the origin of semen in mixed body fluids (stains).

Objectives: To evaluate the forensic application value of an age estimation model based on DNA methylation in eastern Chinese Han population, and to provide a theoretical basis for exploring age estimation models suitable for different detection platforms., Methods: According to the 6 age-related methylation sites in the published blood DNA methylation age estimation models of Chinese Han population, the DNA methylation level of 48 samples was detected by pyrosequencing and next-generation sequencing (NGS). After submitting DNA methylation levels to the age estimation model, the DNA methylation ages were predicted and compared with their real ages., Results: The 6 DNA methylation sites in both detection techniques were age-related, with an R 2 of 0.85 and a median absolute deviation (MAD) of 4.81 years when using pyrosequencing；with an R 2 of 0.84 and MAD of 4.41 years when using NGS., Conclusions: The blood DNA methylation age estimation model can be used under pyrosequencing and multi-purpose regional methylation enrichment sequencing technology based on NGS and it can accurately estimate the age.

With the improvement of DNA methylation detection techniques, studies on age-related methylation sites have found more age-specific ones across tissues, which improves the sensitivity and accuracy of age estimation. In addition, the establishment of various statistical models also provides a new direction for the age estimation of tissues from different sources. This review summarizes the related studies of age estimation based on DNA methylation from the aspects of detection technology, age-related cytosine phosphate guanine site and model selection in recent years.

In forensic physical evidence identification, the accurate identification of the individual origin and their body fluid composition of the biological samples obtained from the crime scene play a critical role in determining the nature of a crime. In recent years, RNA profiling has become one of the fastest developing methods for body fluids identification. Due to the characteristics of tissue or body fluid specific expression, various types of RNA markers have been proven to be promising candidate markers for body fluids identification in previous studies. This review summarizes the research progress of RNA markers in body fluids identification, including the RNA markers that have been effectively verified in current research and their advantages and disadvantages. Meanwhile, this review prospects the application of RNA markers in forensic medicine.

Objectives: To investigate the genetic polymorphism of InDel loci in SifalnDel 45plex system in the Han population in Jiangsu Province and the Mongolian population in Inner Mongolia, and to evaluate the effectiveness of the system in forensic medicine., Methods: SifaInDel 45plex system was used for genotyping in blood samples of 398 unrelated individuals from the above two populations, and allele frequencies and population genetic parameters of the two populations were calculated respectively. Eight intercontinental populations in the gnomAD database were used as reference populations. The genetic distances between the two studied populations and eight reference populations were calculated based on the allele frequencies of 27 autosomal-InDels (A-InDels). The phylogenetic trees and multidimensional scaling (MDS) analysis diagrams were constructed accordingly., Results: Among two studied populations, the 27 A-InDels and 16 X-InDels showed no linkage disequilibrium between each other and the allele frequency distributions were in Hardy-Weinberg equilibrium. The CDP of the 27 A-InDels in two studied populations were all higher than 0.999 999 999 9, and the CPE trio were all less than 0.999 9. The CDP of the 16 X-InDels in Han in Jiangsu and Mongolian in Inner Mongolia female and male samples were 0.999 997 962, 0.999 998 389, and 0.999 818 940, 0.999 856 063, respectively. The CMEC trio were all less than 0.999 9. The results of population genetics showed that the Jiangsu Han nationality, Inner Mongolia Mongolian nationality and East Asian population clustered into one branch, showing closer genetic relationship. The other 7 intercontinental populations clustered into another group. And the above 3 populations displayed distant genetic relationships with the other 7 intercontinental populations., Conclusions: The InDels in the SifaInDel 45plex system have good genetic polymorphism in the two studied populations, which can be used for forensic individual identification or as an effective complement for paternity identification, and to distinguish different intercontinental populations.

Objectives: To study the genetic polymorphism and population genetic parameters of 16 X-STR loci in Xinjiang Uygur population., Methods: The Golden e ye ® DNA identification system 17X was used to amplify 16 X-STR loci in 502 unrelated individuals (251 females and 251 males). The amplified products were detected by 3130 xl genetic analyzer. Allele frequencies and population genetic parameters were analyzed statistically. The genetic distances between Uygur and other 8 populations were calculated. Multidimensional scaling and phylogenetic tree were constructed based on genetic distance., Results: In the 16 X-STR loci, a total of 67 alleles were detected in 502 Xinjiang Uygur unrelated individuals. The allele frequencies ranged from 0.001 3 to 0.572 4. PIC ranged from 0.568 8 to 0.855 3. The cumulative discrimination power in females and males were 0.999 999 999 999 999 and 0.999 999 999 743 071, respectively. The cumulative mean paternity exclusion chance in trios and in duos were 0.999 999 997 791 859 and 0.999 998 989 000 730, respectively. The genetic distance between Uygur population and Kazakh population was closer, and the genetic distance between Uygur and Han population was farther., Conclusions: The 16 X-STR loci are highly polymorphic and suitable for identification in Uygur population, which can provide a powerful supplement for the study of individual identification, paternity identification and population genetics.

Objectives: To estimate the system efficiency of uncle-nephew relationship identification by increasing STR markers and adding reference samples based on the test results of simulated data and real samples, so as to provide references for selecting the appropriate number of STRs and reference samples for uncle-nephew relationship identification., Methods: Five common models of uncle-nephew relationship identification were constructed by adding different reference samples. In each model, the likelihood ratio (LR) for 10 000 pairs of uncle-nephew relationships and 10 000 pairs of unrelated individuals were simulated by detecting 19, 39 or 55 STRs, and the system efficiency at different thresholds was simulated. The samples of the Han population in Zhejiang were collected, and 55 autosomal STRs were obtained by using SiFaSTR TM 23plex kit, Golden e ye ® DNA ID 22NC kit and AGCU 21+1 PCR amplification kit. When 19, 39 and 55 STRs were detected, the LR of each model and system efficiency under different thresholds were calculated and compared with the simulation results., Results: Under the same detection system, the calculated results of simulated data and corresponding true samples were basically consistent. In the same model, there was a positive correlation between the system efficiency of uncle-nephew relationship identification and the number of STRs detected. Moreover, the system efficiency of introducing relatives was higher than identifying only two individuals. The order of preference for the introduction of relatives was the full sibling (or mother) of the uncle and the full sibling (or mother) of the nephew., Conclusions: The system efficiency of uncle-nephew relationship identification could be improved by increasing the number of STRs and introducing known relatives, which would provide the basis for selecting the most appropriate detection system and reference individuals in actual cases.

In recent years, more and more forensic genetics laboratories have begun to apply massively parallel sequencing (MPS) technology, that is, next-generation sequencing (NGS) technology, to detect common forensic genetic markers, including short tandem repeat (STR), single nucleotide polymorphism (SNP), the control region or whole genome of mitochondrial DNA (mtDNA), as well as messenger RNA (mRNA), etc., for forensic practice, such as individual identification, kinship analysis, ancestry inference and body fluid identification. As the most widely used genetic marker in forensic genetics, STR is currently mainly detected by capillary electrophoresis (CE) platform. Compared with CE platform, MPS technology has the advantages of simultaneous detection of a large number of genetic markers, massively parallel detection of samples, the polymorphism of sequence detected by NGS makes STR have the advantages of higher resolution and system efficiency. However, MPS technology is expensive, there is no uniform standard so far, and there are problems such as how to integrate MPS-STR data with the existing CE-STR database. This review summarizes the current status of the application of MPS technology in the detection of STR genetic markers in forensic genetics, puts forward the main problems that need to be solved urgently, and prospects the application prospect of this technology in forensic genetics.

Objectives: To construct a Felis catus STR loci multiplex amplification system and to evaluate its application value by testing the technical performance., Methods: The published Felis catus STR loci data were reviewed and analyzed to select the STR loci and sex identification loci that could be used for Felis catus individual identification and genetic identification. The fluorescent labeling primers were designed to construct the multiplex amplification system. The system was validated for sensitivity, accuracy, balance, stability, species specificity, tissue identity and mixture analysis, and investigated the genetic polymorphisms in 145 unrelated Felis catus samples., Results: Sixteen Felis catus autosomal STR loci and one sex determining region of Y (SRY) were successfully selected, and constructed a multiplex amplification system containing the above loci. The complete profile of all alleles could still be obtained when the amount of DNA template was as low as 0.25 ng. There was no specific amplification peak in other common animal samples. Population genetic surveys showed that total discrimination power (TDP) of the 16 STR loci was 1-3.57×10 -20 , the cumulative probability of exclusion (CPE) was 1-6.35×10 -5 and the cumulative probability of matching was 3.61×10 -20 ., Conclusions: The Felis catus STR multiplex amplification system constructed in this study is highly sensitive, species-specific, and accurate in typing results, which can provide an effective solution for Felis catus species identification, individual identification and kinship identification in the field of forensic science.

Objectives: To evaluate the effects of different pretreatment methods and preservation time on RNA quality of peripheral blood samples, and to optimize the preservation method of peripheral blood samples., Methods: Eight pretreatment methods were used to preprocess the peripheral blood from 3 healthy unrelated individuals and the treated samples were stored at -80 ℃. Total RNA of samples was extracted using Quick-RNA TM Miniprep Plus kit. DNA/RNA Shield TM was added to peripheral blood and total RNA was extracted after preservation at -80 ℃ for 0, 5, 10, 15, 30 and 60 days, respectively. The concentration, purity and integrity of RNA were determined. Statistical analyses were performed by SPSS 22.0 software to compare the differences in RNA yield, purity and integrity among the eight pretreatment methods., Results: In terms of purity, leukocyte pretreated with RNA later TM and directly cryopreservation peripheral blood showed the worst purity. The other six methods showed better purity. In terms of yield, blood cells with DNA/RNA Shield TM came out with the highest yield, followed by peripheral blood with DNA/RNA Shield TM . In terms of integrity, peripheral blood preserved in PAXgene Blood RNA tube method had the best integrity. Except for peripheral blood pretreated with DNA/RNA Shield TM and blood cells pretreated with DNA/RNA shield TM , the other five methods had statistical differences when compared to the method by keeping peripheral blood in PAXgene Blood RNA tube. The purity of RNA stored at six-time gradients ranged from 1.815 to 1.952. With the increase of storage time, RNA yield decreased from 4.516 ng to 1.039 ng, and RNA integrity decreased from 8.533 to 7.150., Conclusions: According to the results of total RNA's yield, purity and integrity, peripheral blood pretreated with DNA/RNA Shield TM was the best pretreatment method. After the pretreatment, samples can be preserved for up to 60 days in low temperature.

Objectives: To evaluate the ability of the ForenSeq TM DNA Signature Prep kit (ForenSeq kit) in analyzing the sequence information of STRs in Zhejiang She ethnic group and its forensic application efficacy., Methods: A total of 50 Zhejiang She ethnic group samples were sequenced with the ForenSeq kit on the MiSeq FGx platform. The data was analyzed using ForenSeq TM universal analysis software to obtain the motif structure and flank regions of the 58 STRs, then compared with PCR-CE typing results to test the consistency. At last, the allele frequency and population genetic parameters were calculated., Results: A total of 448 sequence polymorphic alleles were detected in 50 samples of Zhejiang She ethnic group. Compared with fragment length polymorphism detected by PCR-CE, 82 alleles were increased by MPS detection based on ForenSeq kit, and 7 SNPs variation were detected in the flanking regions of 6 loci. The 22 male individuals were genotyped, and total 19 haplotypes were detected in 24 Y chromosome STRs of these 22 males. The cumulative discrimination power of the 27 autosomal STRs was 1-8.87×10 -30 , the cumulative probability of exclusion of duo-testing was 0.999 999 962 640 657, the cumulative probability of exclusion of trios-testing was 0.999 999 999 999 633., Conclusions: Based on MPS typing technology, using the ForenSeq kit greatly improves the detection efficiency. In addition, the 58 STRs have good genetic polymorphisms in Zhejiang She ethnic group, which are suitable for individual identification and paternity identification in forensic application.

Abstract: Objective To study the genetic polymorphism of whole mitochondrial DNA （mtDNA） genomes in She population in Zhejiang and to explore the maternal genetic structure of the She population. Methods Whole mtDNA genomes of 231 unrelated individuals from She population in Zhejiang Province were sequenced. The number of mutations and population genetics parameters such as, the haplotype diversity （HD）, discrimination power （DP）, and random match probabilities （RMP） were analyzed. The mtDNA haplogroups of Zhejiang She population were classified, and the maternal genetic relationships between She and nine other Chinese populations were estimated. Results In 231 Zhejiang She samples, 8 507 mutations （702 types） were observed and the samples were classified into 94 haplogroups. The HD, DP and RMP values were 0.998 6, 0.994 2 and 0.005 8, respectively. The lowest genetic differentiation degree （ F st =0.006 89） was detected between Zhejiang She population and southern Han population. Principal component analysis （PCA） and median-joining network analysis showed that the genetic distance of Zhejiang She population with Guangxi Yao, Yunnan Dai and Southern Han populations was relatively close, but the population still had some unique genetic characteristics. Conclusion The whole mtDNA genomes are highly polymorphic in Zhejiang She population. The Zhejiang She population contains complex and diverse genetic components and has a relatively close maternal genetic relationship with Guangxi Yao, Yunnan Dai and Southern Han populations. Meanwhile, Zhejiang She population has kept its unique maternal genetic components., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Abstract: Objective To assess the feasibility of the rbcL sequence of chloroplast DNA as a genetic marker to identify Cannabis sativa L. Methods The rbcL sequences in 62 Cannabis sativa L. samples, 10 Humulus lupulus samples and 10 Humulus scandens DNA samples were detected, and 96 rbcL sequences of the Cannabaceae family were downloaded from Genbank. Sequence alignment was performed by MEGA X software, the intraspecific and interspecific Kimura-2-Parameter （K2P） genetic distances were calculated, and the system clustering tree was constructed. Results The rbcL sequence length acquired by sequencing of Cannabis sativa L. and Humulus scandens were 617 bp and 649 bp, respectively, and two haplotypes of Cannabis sativa L. were observed in the samples. The BLAST similarity search results showed that the highest similarity between the sequences acquired by sequencing and Cannabis sativa L. rbcL sequences available from Genbank was 100%. The genetic distance analysis showed that the maximum intraspecific genetic distance （0.004 9） of Cannabis sativa L. was less than the minimum interspecific genetic distance （0.012 9）. The results of median-joining network and system clustering tree analysis showed that Cannabis sativa L. and other members of the Cannabaceae family were located in different branches. Conclusion The rbcL sequence could be used as a DNA barcode for identifying Cannabis sativa L., and combined with comparative analysis of the rbcL sequence and system cluster analysis could be a reliable and effective detection method for Cannabis sativa L. identification in forensic investigation., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Abstract: The paternal inheritance characteristics of Y chromosome have been widely used in the forensic genetics field to detect the genetic markers in the non-recombining block, and used in the studies such as, genetic relationship identification, mixed stain detection, pedigree screen and ethnicity determination. At present, capillary electrophoresis is still the most common detection technology. The commercial detection kits and data analysis and processing system based on this technology are very mature. However, the disadvantages of traditional detection technology have gradually appeared with the rapid growth of bio-information amount, which promotes the renewal of forensic DNA typing technology. In recent years, next generation sequencing （NGS） technology has developed rapidly. This technology has been applied to various fields including forensic genetics and has provided new techniques for the detection of Y chromosome genetic markers. This article describes the current situation and application prospects of the NGS technology in forensic Y chromosome genetic markers detection in order to provide new ideas for future judicial practice., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Abstract: Recently, researches on copy number variation （CNV） have extended to every field, such as etiological exploration and precise treatment of complex diseases, as well as genetic breeding and evolution. The unique genetic characteristics of CNV have made people gradually believe that it could be used as a biological genetic marker to solve related problems. With the development of detection technology, application of CNV in forensic medicine will increase gradually. In this paper, the concept and development of CNV, as well as its application in forensic medicine are summarized, to provide new ideas for the practical application of CNV in the future., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Abstract: Age estimation is of great significance in the fields of criminal investigation and forensic identification. It can provide the age information of individuals to judicial departments to facilitate the development of judicial work. In recent years, age estimation methods expanded from the morphological level to the molecular biology level. With the rapid development of epigenetics represented by DNA methylation, and the advancement of DNA methylation detection technology together with the detection platform, many age estimation methods based on DNA methylation biomarkers, or using several biological fluids, such as blood, blood stains, saliva, semen stains, etc. are developed. Currently, researches related to age estimation based on DNA methylation are relatively widely carried out. This paper summarizes the researches on age estimation based on DNA methylation, in order to provide references for related studies and forensic applications., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Abstract: The new Standardization Law, implemented in 2018, has added a standard post-implementation evaluation system, aiming to continuously improve the quality of standards through post-implementation evaluation. Standards in the forensic science field are closely related to accreditation activities. Forensic science standards are not only the criteria on which accreditation activities are carried out, but also one of the key contents of the inspection of forensic science institutions in accreditation activities. Since 2018, the certification and accreditation policies in the forensic science field have also been changed, which has brought impacts on the construction of a standard system based on accreditation.This paper analyzes the standard data from China National Accreditation Center from Conformity Assessment on forensic clinical identification accreditation assessment. It points out that the current coverage of laboratory accreditation activities is limited, the development in different provinces is unbalanced, and there is overlap and crossover in the standards in use. It is emphasized that the construction of the national forensic science standardization technical committee, the improvement of the forensic science standard system, the establishment of the standard implementation evaluation index system, and promotion of the coordination of standards, and the certifications and accreditations should be accelerated, in order to continue to promote the standardization and accreditation activities in the field of forensic science., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Abstract: Objective To investigate the application of the comprehensive use of multiple genetic markers in full and half sibling relationship testing through the identification of a case of suspected sibling relationship. Methods Genomic DNA were extracted from bloodstain samples from 4 subjects （ZHANG-1, ZHANG-2, male; ZHANG-3, ZHANG-4, female）. Autosomal STR loci, X-STR, Y-STR loci and polymorphisms of mtDNA HV-Ⅰ and Ⅱwere genotyped by EX20 STR kit, X19 kit, Data Y24 STR kit, and Sanger sequencing, respectively. Results According to autosomal STR based IBS scoring results, full sibling relationships were indicated among ZHANG-2, ZHANG-3 and ZHANG-4, but those were not indicated between ZHANG-1 and ZHANG-2 or ZHANG-3 or ZHANG-4. According to autosomal STR based FSI and HSI, with ITO method and discriminant function method, full sibling relationships among ZHANG-2, ZHANG-3 and ZHANG-4 were indicated, and half sibling relationships between ZHANG-1 and ZHANG-2 or ZHANG-3 or ZHANG-4 were also indicated. X-STR and mtDNA sequencing results showed that all the 4 samples came from a same maternal line, and Y-STR results showed that ZHANG-1 and ZHANG-2 did not come from a same paternal line, which supported the half sibling relationship between ZHANG-1 and ZHANG-2 or ZHANG-3 or ZHANG-4, verified by parental genotype reconstruction based on autosomal STR genotyping. Conclusion For the identification of sibling relationships, it is effective to have reliable results with the mutual verification and support of multiple genetic markers （autosomal STR, sex chromosomal STR and mtDNA sequence） and calculations （IBS, ITO, discriminant function method and family reconstruction）., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Abstract: Objective To evaluate the efficiency of REPLI-g® Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g® Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. Golden e ye® DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g® Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony® DNA Investigator® Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g® Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g® Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Objectives: To analyze the literature on forensic sciences indexed in Science Citation Index Expanded （SCIE） in recent 10 years, and to understand the research status, characteristics and trends in the field of forensic sciences., Methods: Literature on forensic sciences from 2008 to 2017 in Web of Science （WoS） was retrieved. The documents number and geographical distribution, document types, source titles, organizations, research areas, authors, funding agencies, and the high cited articles were detected. The impact factors （IF） of journals were retrieved in Journal Citation Reports （JCR）. The data were analyzed with descriptive statistics., Results: From 2008 to 2017, there were 21 001 documents on forensic sciences in SCIE. The main document type was articles, with English as the major language. With regards to research areas, pathology has the largest number of papers worldwide, and genetics and heredity has the largest number of publications in mainland China. Among the 18 journals where the documents was published, Forensic Science International ranks the first on publication count, and Forensic Science International Genetics has the highest IF （5.637） in the JCR 2017. In 2017, the number of papers from mainland China increased by 48.50% compared with 2016, which was higher than the global increase （32.63%） and the top-5 countries in terms of number of publications （the US, Germany, the UK, Australia, Italy）. The average document count per organization is 1.98 worldwide and 1.17 in mainland China, respectively. The publication number per author is 0.53 worldwide and 0.36 in mainland China, respectively. Around 28.17% of the publications were funded, with National Natural Science Foundation of China （NSFC） as the Top 1 funding agency （192 papers）. Among the documents with citations, the most cited publication has been cited for 366 times., Conclusions: The yearly numbers of publications on forensic sciences are increasing during recent 10 years. Focusing on the mainland China, there would be more high-quality papers with the steady funding of NSFC., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Genetic markers in forensic DNA typing experienced the variable number of tandem repeats （VNTR） sequences and the short tandem repeats （STR） sequences. With the emerge of sequencing technology, the third generation of genetic markers were found out, which usually have two alleles including single nucleotide polymorphism （SNP） and insertion/deletion （InDel）, also known as biallelic genetic markers. Because of the insertions or deletions of DNA fragments, InDel genetic marker reveals DNA fragment length polymorphism and widely distributes across the whole genome. InDel genetic marker is numerous and has the characteristics of STR and SNP genetic markers, which has been applied in the fields of genetics and anthropology. This review focuses on the research progress of InDel genetic marker in forensic science, aiming to review and summarize the main research findings in recent years and provide clues for future researches., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Objectives: To derive the probability equation given by STR allele frequencies of identity by state （IBS） score shared by unrelated individual pairs., Methods: By comparing the STR genotypes of two unrelated individuals, three mutually exclusive combinations could be obtained： （1） sharing 2 identical alleles, a ₂=1, otherwise a ₂=0; （2） sharing 1 identical allele, a ₁=1, otherwise a ₁=0; （3） sharing 0 identical allele, a ₀=1, otherwise a ₀=0. And the IBS score of the one STR locus in this unrelated individual pair could be given by the formula： ibs =2 a ₂+ a ₁. The probability of a ₂=1 （ p ₂）, a ₁=1 （ p ₁） and a ₀=1 （ p ₀） were derived and expressed in powers of the allele frequencies. Subsequently, for a genotyping system including n independent STR loci, the characteristics of binomial distribution of IBS score shared by a pair of unrelated individuals could be given by p ₂ l and p ₁ l （ l =1, 2, …, n ）., Results: All the general equations of p ₂, p ₁ and p ₀ were derived from the basic conceptions of a ₂, a ₁ and a ₀, respectively. Given f i （ i =1, 2, …, m ） as the ith allele frequency of a STR locus, the general equations of p ₂, p ₁ and p ₀ could be respectively expressed in powers of f i ： [Formula: see text],[Formula: see text] and [Formula: see text]. The sum of p ₂, p ₁ and p ₀ must be equal to 1. Then, the binomial distribution of IBS score shared by unrelated individual pairs genotyped with n independently STR loci could be written by： IBS ~B（2 n , π ）, and the general probability, π, could be given by the formula： [Formula: see text]., Conclusions: In the biological full sibling identification, the probability of null hypothesis corresponding to any specific IBS score can be directly calculated by the general equations presented in this study, which is the basement of the evidence explanation., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Individual identification plays an import role in the practice of forensic medicine, and often provides crucial evidence for the analysis and detection of criminal cases. However, for individual identification in complex situations, such as monozygotic （MZ） twins assumed to be genetically identical, it is impossible to distinguish one from the other by using traditional forensic DNA typing system. Therefore, how to discriminate the MZ twins has become and will continue to be one of the difficult problems in forensic field. This paper summarized the genetic and epigenetic changes recently identified in MZ pairs, which might provide a new insight to forensic discrimination of MZ twins., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Objectives: To select a Y-STR marker system with strong haplotype identification ability, appropriate mutation rate and high compatibility and to assess its forensic application., Methods: The 24 Y-STR loci were tested by self-built fluorescent multiplex system, and the forensic assessment was conducted by 139 pairs of father-son samples collected in Jinan, Shandong province., Results: Totally 176 alleles were identified among the 24 Y-STR loci in the sample of 139 unrelated individuals labeled with father, and the gene diversity （GD） distributed between 0.083 7 （ DYS645 ）-0.966 9 （ DYS385a/b ）. According to the 24 Y-STR loci, 139 different haplotypes were detected from 139 unrelated male individuals labeled with father in Han population of Shandong province and with no shared haplotype observed. The overall haplotype diversity （HD） was 1 and the discrimination capacity （DC） was 1. A total of 5 one-step mutations events were observed among the 24 Y-STR loci in 139 pairs of father-son. The average mutation rate was 0.001 5 [95% CI （0.000 5, 0.003 5）]., Conclusions: The system of 24 Y-STR loci shows a strong individual recognition ability and low mutation rate in the population in Jinan, Shandong province, and it has good application value in forensic science., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Objectives: To investigate the genetic polymorphism of 21 autosomal STR loci and DYS391 locus of SiFaSTR™ 23plex DNA ID system in Han population of eastern China and to evaluate its application value in forensic science., Methods: Typing test of 2 000 unrelated individuals was performed using SiFaSTR™ 23plex DNA ID system. The population genetic parameters of STR loci were statistically analysed. A total of 3 198 parentage confirmed cases were detected with that system and the mutation conditions were observed in 21 autosomal STR loci., Results: All the 21 autosomal STR loci showed no significant departure from Hardy-Weinberg equilibrium （ P >0.05）. The Ho ranged from 0.617 5 to 0.927 0. The DP ranged from 0.796 4 to 0.986 9, as well as the PIC distributed from 0.561 1 to 0.912 3. The CDP was 0.999 999 999 999 999. The CPE duo was 0.999 997 431 701 961, while CPE trio was 0.999 999 999 654 865. Five alleles were detected in DYS391 locus, with the allele frequency from 0.004 0 to 0.729 0, and GD was 0.418 9. Except D13S317 and D10S1248 , seventy-six mutation events were observed at the rest nineteen autosomal STR loci. Among them, seventy-five （98.68%） were one step mutation, and only one （1.32%） was three steps mutation. The mutation rate ranged from 0.246 5×10⁻³ to 2.711 4×10⁻³, and the averaged mutation rate was 0.892 1×10⁻³ （95% CI： 0.70×10⁻³-1.10×10⁻³）. In 33 trio mutation cases, the proportion of the paternal mutation and the maternal mutation was 2.09:1., Conclusions: The involved STRs are highly polymorphic in Eastern Han population with acceptable mutation rates by the SiFaSTR™ 23plex DNA ID system, which is suitable for paternity testing and individual identification., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Objectives: To establish a query table of IBS critical value and identification power for the detection systems with different numbers of STR loci under different false judgment standards., Methods: Samples of 267 pairs of full siblings and 360 pairs of unrelated individuals were collected and 19 autosomal STR loci were genotyped by Golden e ye™ 20A system. The full siblings were determined using IBS scoring method according to the 'Regulation for biological full sibling testing'. The critical values and identification power for the detection systems with different numbers of STR loci under different false judgment standards were calculated by theoretical methods., Results: According to the formal IBS scoring criteria, the identification power of full siblings and unrelated individuals was 0.764 0 and the rate of false judgment was 0. The results of theoretical calculation were consistent with that of sample observation. The query table of IBS critical value for identification of full sibling detection systems with different numbers of STR loci was successfully established., Conclusions: The IBS scoring method defined by the regulation has high detection efficiency and low false judgment rate, which provides a relatively conservative result. The query table of IBS critical value for identification of full sibling detection systems with different numbers of STR loci provides an important reference data for the result judgment of full sibling testing and owns a considerable practical value., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine)

The rapid development of second generation sequencing （SGS） within the past few years has led to the increasement of data throughput and read length while at the same time brought down substantially the sequencing cost. This made new breakthrough in the area of biology and ushered the forensic genetics into a new era. Based on the history of sequencing application in forensic genetics, this paper reviews the importance of sequencing technologies for genetic marker detection. The application status and potential of SGS in forensic genetics are discussed based on the already explored SGS platforms of Roche, Illumina and Life Technologies. With these platforms, DNA markers （SNP, STR）, RNA markers （mRNA, microRNA） and whole mtDNA can be sequenced. However, development and validation of application kits, maturation of analysis software, connection to the existing databases and the possible ethical issues occurred with big data will be the key factors that determine whether this technology can substitute or supplement PCR-CE, the mature technology, and be widely used for cases detection., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

RNA has received more attention in the field of forensic medicine and the development of the new biological markers based on RNA shows great significance in the analysis of complex cases. circular RNA (circRNA) is a kind of non-coding RNA which is widely reported recently. Although the regulatory mechanisms of generation and expression are not fully clear, the existing research indicates that circRNA has important biological functions. CircRNA has a cell-type-specific expression with great stability and a high expression level, which makes it meaningful in forensic applications potentially. In this paper, the research progress, the generation and regulation of circRNA as well as its biological characteristics and functions are summarized, which will provide references for related studies and forensic applications.

Species identification of biological samples is widely used in such fields as forensic science and food industry. A variety of accurate and reliable methods have been developed in recent years. The current review shows common target genes and screening criteria suitable for species identification, and described various DNA-based molecular biology methods about species identification. Additionally, it discusses the future development of species identification combined with real-time PCR and sequencing technologies.

Objective: To evaluate the forensic application value of 30 insertion/deletion (InDel) loci included in Investigator DIPplex Kit in Han and She nationalities of Eastern China., Methods: A total of 565 unrelated individuals in Han nationality and 119 ones in She nationality of Eastern China were investigated using Investigator DIPplex Kit. Allele frequencies, population genetics parameters of the 30 InDel loci were statistically calculated., Results: In Han nationality, the mean Ho was 0.413 3, the mean DP was 0.551 1, the mean PIC was 0.320 0. And in She nationality, the mean Ho was 0.389 6, the mean DP was 0.543 3, the mean PIC was 0.310 0. No deviation from Hardy-Weinberg equilibrium was observed in Han and She nationalities (P > 0.05)., Conclusion: The 30 loci in Investigator DIPplex Kit show good genetic diversity in Han and She nationalities, and could be used as a supplemental tool for some special paternity cases.

Objective: To investigate Insertion/Deletion (InDel) polymorphism on the X chromosome and to screen 18 InDel loci for the Chinese Han population as a forensic DNA typing system auxiliary., Methods: Eighteen X-InDel markers were selected using the Human Genome Browser and dbSNP database. Multiplex PCR primer pairs of selected X-InDel markers were designed using Primer 3 software and divided into 3 groups according to the amplified fragment length, labeled by FAM, HEX and TAMRA fluorescence dye, respectively. The population genetics research and comparative analysis of Chinese Han nationality and 4 main minorities, the Hui, Wei, Mongol, and Tibetan nationalities, were investigated with the system., Results: A new multiplex genotyping system, named InDel X-18PLEX, was successfully developed and validated, consisted of 18 X-InDel markers on the X chromosome and 1 Amelogenin gender marker. No deviation from Hardy-Weinberg equilibrium expectations was detected in the distribution of genotypes in the 5 investigated ethnic groups. However, there was significant difference between their distributions. From the investigation of Han nationality, high female (0.9999994) and male (0.999 88) overall discrimination power values were obtained, as well as high overall mean exclusion chance values in trios (0.999 992) and in duos (0.99)., Conclusion: InDel X-18PLEX meets the requirements as a forensic DNA complementary kit, providing effective supplementary analytical tools for difficult cases.

Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link between sample donors and actual criminal acts. However, the conventional body fluid identification methods are prone to various limitations, such as time consumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Recently, the analysis of cell-specific messenger RNA expression (mRNA profiling) has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group (EDNAP) in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profiling, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibility with current DNA extraction and analysis procedure. In the current review, we provided an overview of the present knowledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possible practical application to forensic casework.

As forensic DNA typing experienced three generations of genetic marker researching stage, short tandem repeat (STR) has been widely used in forensic identification as a mature tool. Further exploration of the human genome led to the discovery of polymorphism markers of single nucleotide polymorphism (SNP) and Insertion/Deletion (InDel). InDel, which combines the desirable characteristics of previous genetic markers as a new type of genetic marker, has got extensive concern in fields like medical molecular biology and forensic biology. This paper generally reviews the history of research and the corresponding results of InDel along the line of time axis as well as the different aims of these research focusing on the progress in the multiple amplification system with several InDel as the genetic marker (autosomal or X chromosome) in forensic biology and anthropology. Finally, the direction of research in this field and the problems to be solved have been put forward.

Objective: To investigate the genetic data of 12 autosomal STR loci included in Investigator HDplex kit and to evaluate its forensic application in Han nationality of Eastern China., Methods: A total of 484 unrelated healthy individuals in Han nationality of Eastern China were investigated with Investigator HDplex kit. Allele frequencies, population genetics parameters and linkage disequilibrium information of the 12 autosomal STR loci were statistically analyzed., Results: No deviations from Hardy-Weinberg equilibrium were detected and all loci were independent form each other within the studied 484 unrelated healthy individuals. DP values of the 12 autosomal STR loci were all above 0.8, and CDP was 0.999 999 999 92. The cumulative probability of paternity exclusion in duo and in trio were 0.999 82 and 0.999 998 6, respectively., Conclusion: Investigator HDplex kit with 12 highly polymorphic STR loci in Han nationality of Eastern China could be used effectively for forensic DNA genotyping.

Objective: To derive the formulae for likelihood ratio calculation in discriminating full sibling from half sibling with single-parent participation or without parent participation., Methods: Null hypothesis and alternative hypothesis were established for discriminating full sibling from half sibling in two circumstances: two children with single-parent and without parent participation. Conditional probabilities of the genetic evidentiary under null and alternative hypotheses were calculated according to the Bayesian theory. The likelihood ratios were established with the conditional probability under alternative hypothesis division that under null hypothesis, followed with simplification. All the formulae were validated in a real case., Results: While mother or fathers' genetic information available in differentiating full sibling from half sibling, 14 different genotype combinations could be shared by the two detected children at a given locus and the likelihood ratio could be calculated with 5 different formulae respectively. While both parents' genetic information unavailable, 11 different genotype combinations could be shared and the likelihood ratio could be calculated with 7 different formulae respectively. It was validated in a real case that the power of the likelihood ratio method developed for discriminating full sibling from half sibling with single-parent participation was higher than that of the ratio of full sibling index over half sibling index., Conclusion: The formulae of likelihood ratio developed are useful for discriminating full sibling from half sibling with single-parent participation or without parent participation.

Objective: To develop a PCR-based X-STR kit for typing of 16 X-STR loci and investigate the polymorphisms of the X-STR markers., Methods: Sixteen STR loci (GATA 165B12, DXS101, GATA 172D05, HPRTB, DXS981, DXS8378, DXS6795, GATA 31E08, DXS6809, DXS6803, DXS9902, DXS6807, DXS7423, DXS7133, DXS6810 and DXS7132) located on X chromosome were selected. The primers for multiplex PCR were designed by Primer Premier 5.0 software and labeled by four fluorescences (FAM, HEX, TAMRA and ROX). The developed multiplex PCR system was used for investigating the polymorphisms of the X-STR markers in Han populations., Results: The 16-plex amplification system named IDtyper X-16 was successfully developed and validated. Among the 16 X-STR loci, DXS7133 and DXS7423 were found to be moderately polymorphic and the other 14 X-STR markers were highly polymorphic (P1C > 0.5, H > 0.5). The cumulative discrimination power in females and in males were 0.999 999 999 999 97 and 0.999 999 993 respectively in Han population. The combined power of exclusion in trios and in duos were 0.999 999 93 and 0.999990, respectively., Conclusion: The IDtyper X-16 kit is highly valuable in forensic science and is suitable for paternity testing in disputed cases.

Objective: To establish universal algorithms for commonly used kinship indices between two individuals., Methods: Based on the formulas of paternity index in duos(PID), full sibling index(FSI), half sibling index (HSI), avuncular index (AI), grandparental index (GI) and first cousin index (CI1st) deduced from ITO method, the common factors, 1 plus reciprocal of the frequency of the allele with identity by state between the two individuals, shared in these formulas were abstracted with induction method, following with reconstruction of these formulas with the common factor and the coefficient of relationship (r)., Results: A universal algorithm for PI(D), HSI, AI, GI and CI1st, was developed with the common factor and r value according to the heterozygosity of the two individuals. Meanwhile, a group of two formulas for FSI calculation was also established according to the individuals' heterozygosity., Conclusion: The universal algorithms for the 6 types of kinship indices are practical in corresponding kinship determination and the batch arithmetic operation with the universal algorithms can be easily programmed.

Objective: To screen a panel of SNP loci on X chromosome (X-SNP loci) that is informative in Chinese Han population and evaluate its potential value in forensic identification., Methods: Sixty-seven candidate X-SNP loci were selected according to the information on dbSNP and HapMap. Genomic DNA extracted from blood samples of 428 unrelated Chinese Han individuals were analyzed through multiplex amplification followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry, and allele frequencies of the 67 X-SNP loci were calculated. In view of the population data and situation of linkage disequilibrium, X-SNP markers promising in forensic identification were sorted out., Results: Population data of the 67 X-SNP loci were obtained. Except rs12849634, no deviations from Hardy-Weinberg equilibrium could be found. Of the rest 66 X-SNP loci, two loci (rs1229078 and rs1544545) were found to be low informative (minor allele frequency < 0.3). Closely linked alleles were observed at six pairs of X-SNP loci and slightly linked alleles were observed at two pairs of X-SNP loci. Fifty-two X-SNP loci showing independent inheritance and high polymorphisms were finally selected. The markers were promising in forensic identification. CPE in trio and duo cases were 0.999 999 999 96 and 0.999 999 5, respectively. CDP in female and male populations were 0.999 999 999 999 999 999 999 84 and 0.999 999 999 999 999 31, respectively., Conclusion: A panel of 52 informative X-SNP loci showing independent inheritance is selected. These markers meet the needs of individual identification and relationship testing in judicially disputed cases.

Objective: To investigate the genetic data of the 12 X-STR included in Investigator Argus X-12 kit and to evaluate the forensic application in Han population from East China. 9: By detecting 309 unrelated individuals with Investigator Argus X-12 kit, allele frequencies, population genetics parameters and the information of linkage disequilibrium of the 12 X-STR were analyzed by statistics and were compared with available data of other Han populations from different regions., Results: No deviations from Hardy-Weinberg equilibrium were detected. Except loci of DXS10103 and DXS10101 linked closely, all other loci were independent while HET exceed 0.5 and PIC all above 0.4. Distributions of allele frequencies of all loci were not significant statistically except for locus of DXS10146 in Han population from Guangdong., Conclusion: Loci of Investigator Argus X-12 kit were highly polymorphic in Han population from East China, which is suitable for forensic application in paternity testing and individual identification.

Objective: To evaluate the potential usefulness of DNA methylation in individual discrimination of monozygotic twins by investigating the differences of DNA methylation profiles in monozygotic twins' blood samples., Methods: Blood samples from 22 pairs of monozygotic twins were obtained with informed consent. Genomic DNA extracts were bisulfite treated followed by detection with Infinium HumanMethylation27 BeadChip Assays(Illumina, USA). Epigenetic distances between each pair of monozygotic twins and each pair of unrelated individuals of same gender were calculated with Euclidean distance algorithms. Distribution of epigenetic distance in monozygotic twin group was statistically compared with that in unrelated individuals., Results: Difference of epigenetic distance between male and female pairs was not statistically significant in unrelated individual group or in monozygotic twin group (P = 0.0695 and 0.4825, respectively). Epigenetic distance of monozygotic twins was significantly lower than that of unrelated individual pair of same gender (Median: 6.02 vs 7.20, P = 0.0002). However, all the epigenetic distance in monozygotic twin group or in unrelated individuals were significantly higher than 4.00 (P < 0.000 1)., Conclusion: DNA methylation profiles of monozygotic twin's blood samples were significantly different with each other, which was similar to that in unrelated individuals of same gender. These results indicated that DNA methylation was a useful biomarker in individual discrimination of monozygotic twins.

Objective: To investigate the criterion for source identification of gastrointestinal tumor based on the number of identical allele (IAn) and the number of matched STR locus with 2 identical alleles (A2) in Identifiler system., Methods: One hundred and five pairs of gastrointestinal tumor samples and homologous normal samples (TN group) were genotyped with Identifiler system. The numbers of STR locus with genotypic alteration (STRGA) in each tumor were determined by comparing the genotype of the matched STR loci in each pair of samples. According to the limited distribution of IAn and A2, 16 different values of IAn was substituted into the published discriminant functions to obtain the cut-off values of IAn and A2 for source identification of tumor sample. Indices including sensitivity (SEN), specificity (SPE), accuracy (AC), positive predictive value (PPV) and negative predictive value (NPV) for distinguishing tumor from an unrelated individual or a full sibling of the patient were calculated. Concordance of the identification results based on the determined criteria and the definite facts were statistically tested with Kappa index., Results: The total frequency of STRGA was 5.46%. There were 31.43% of the 105 tumor samples carried at least one STR locus with STRGA mutation. According to the Fisher discrimination rules, criteria I (IAn>or=23 and A2>or=8) and criteriall (IAn>or=26 and A2>or=11) meet the requirements of distinguishing tumor sample from an unrelated individual or a full sibling of the patient with tumor, respectively. SEN=0.971 0, PPV=1.000 0, PPV=0.891 9 and Kappa=0.923 5, when the criteria were used to determine the specified relatives., Conclusion: Criteria I and criteria II were powerful for distinguishing tumor sample from an unrelated individual or a full sibling of the patient with tumor, respectively, when the Identifiler system was adopted for source identification of gastrointestinal tumor sample.

Objective: To investigate the criteria of the number of identical allele (IAn) and the number of matched STR locus with 2 identical alleles (A2) for full sibling (FS) determination with Identifiler system., Methods: According to the limited distribution of IAn. and A2, all of the 31 potential values of IAn. were substituted into the published discriminant functions to obtain the cut-off values of IAn and A2 for FS determination, and then 4 different criteria were determined to distinguish 280 FS pairs from 2283 individual pairs, respectively, which had been genotyped with Identifiler system. Cumulative full sibling index (CFSI) of the samples were calculated with ITO method, and 4 different criteria of CFSI (>1, > or =5, > or =20 and > or =100) were also utilized to determinate FS, respectively. Indices including sensitivity (SEN), specificity (SPE), accuracy(AC), positive predictive value(PPV) and negative predictive value (NPV) of the 8 different criteria for FS determination were calculated, respectively. Concordance of FS determination between the criteria based on IAn and A2 and that of CFSI were statistically tested with Kappa index., Results: All the individual pairs, which meet the requirement of (I) IAn > or =15 and A2 > or =4, or (II) IAn > or =16 and A2 > or =3, or (III) IAn > or = 17 and A2 > or =3, or (IV) IAn > or =18 and A2 > or =3, could been concluded as FS. AC, SPE and NPV of the 4 criteria mentioned above and the 4 criteria of CFSI were all over 0.9500 in FS determination. Indices between criterion II and CFSI > or =5, criterion III and CFSI > or =20, criterion IV and CFSI > or =100 were similar with each other and the Kappa indexes of the 3 groups were 0.9049, 0.9204 and 0.9083, respectively. PPV and NPV of criterion III and CFSI > or =20 were all over 0.9500., Conclusion: The criterion of IAn > or =17 and A2 > or =3 was feasible and efficient for FS determination with Identifiler system, power of which was similar with the criterion of CFSI > or =20.

Objective: To study the effect of urinary trypsin inhibitor (UTI) on STR genotyping with urinary samples., Methods: Midstream urine samples of 5 male and 5 female volunteers were collected respectively, sub-packaged, added with different concentration of UTI and stored at -80 degrees C. Genomic DNA was extracted from those urinary samples, of which STR profiles were genotyped with IdentifilerTM kit at 8 different time points. Results of genotyping in urinary samples were compared with those of the homogenous blood control samples and the successful rate of genotyping in different group of urinary samples treated with UTI was determined., Results: Fifteen STR loci included in Identifiler system were all detected in control blood samples and urinary samples stored for 1 day. STR locus loss was observed and all 15 STR loci disappeared in female urinary samples untreated with UTI while those storage periods prolonged to 3 and 9 days, respectively. However, all 15 STR loci could be detected in female urinary samples treated with UTI and stored for as long as 9 days. No STR loci could be detected in male urinary samples preserved without UTI for 7 days while 9 STR loci detected preserved with UTI for 9 days. There was no significant difference among the average detection ratios of STR loci in female urinary samples treated with UTI at concentrations of 0.2, 0.4 or 0.6 microg/mL and stored for 30 days, mean of which was as high as 0.8400 +/- 0.0423, statistically higher than that in male urinary samples (0.1600 +/- 0.0423)., Conclusion: Detection rate of STR loci in urinary samples preserved with UTI was increased significantly, which results in prolonging the storage periods of urinary samples for personal identification.

Objective: To develop a multiplex PCR system, using insertion/deletion (InDel) polymorphism markers, for forensic DNA identification among Han, Hui, Uighur, Mongolian and Tibetan populations in China., Methods: Highly polymorphic InDel markers from human autosomes were selected using the Human Genome Browser in Galaxy system and dbSNP database. Multiplex PCR primer pairs of selected InDel markers were designed using Primer 3 software. The multiplex PCR system was developed using a five fluorescence dye labeling system. Genetic polymorphisms of selected InDel markers were investigated using the multiplex PCR system among five populations in China., Results: A new multiplex genotyping system, named InDel&#95;typer30, was successfully developed and validated in this study. The InDel&#95;typer30 system consisted of 30 highly polymorphic InDel markers and 1 Amelogenin gender marker. The average expected heterozygosity of the 30 InDel markers was 0.464, 0.460, 0.453, 0.466 and 0.469 for the Han, Hui, Uighur, Mongolian and Tibetan populations, respectively. The average discrimination power was 0.595, 0.585, 0.586, 0.589 and 0.595 for the Han, Hui, Uighur, Mongolian and Tibetan populations, respectively. The cumulative discrimination power (CDP) were all above 0.999 999 999 996 for the 5 populations., Conclusion: InDel&#95;typer30 was a useful forensic DNA identification tool for human identification among Han, Hui, Uighur, Mongolian and Tibetan populations in China.

Objective: Determination strategies for half sibling sharing a same mother were investigated through the detection of autosomal and X-chromosomal STR (X-STR) loci and polymorphisms on hypervariable (HV) region of mitochondrial DNA (mtDNA)., Methods: Genomic DNA were extracted from blood stain samples of the 3 full siblings and one dubious half sibling sharing the same mother with them. Fifteen autosomal STR loci were genotyped by Sinofiler kit, and 19 X-STR loci were genotyped by Mentype Argus X-8 kit and 16 plex in-house system. Polymorphisms of mtDNA HV-I and HV-II were also detected with sequencing technology., Results: Full sibling relationship between the dubious half sibling and each of the 3 full siblings were excluded based on the results of autosomal STR genotyping and calculation of full sibling index (FSI) and half sibling index (HIS). Results of sequencing for mtDNA HV-I and HV-II showed that all of the 4 samples came from a same maternal line. X-STR genotyping results determined that the dubious half sibling shared a same mother with the 3 full siblings., Conclusion: It is reliable to combine three different genotyping technologies including autosomal STR, X-STR and sequencing of mtDNA HV-I and HV-II for determination of half sibling sharing a same mother.