Application Progress of Massively Parallel Sequencing Technology in STR Genetic Marker Detection / 法医学杂志

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.

Genetic Polymorphism of 16 X-STR Loci in Xinjiang Uygur Population / 法医学杂志

OBJECTIVES@#To study the genetic polymorphism and population genetic parameters of 16 X-STR loci in Xinjiang Uygur population.@*METHODS@#The Goldeneye® 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 3130xl 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.

Forensic Application of ForenSeqTM DNA Signature Prep Kit in Zhengjiang She Ethnic Group / 法医学杂志

OBJECTIVES@#To evaluate the ability of the ForenSeqTM 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 ForenSeqTM 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.

Identification of Cannabis Sativa L. Based on rbcL Sequence / 法医学杂志

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.

Mitochondrial DNA Polymorphism in Zhejiang She Population Based on Next Generation Sequencing / 法医学杂志

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 （Fst=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.

Research Progress on InDel Genetic Marker in Forensic Science / 法医学杂志

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.

Review of Second Generation Sequencing and Its Application in Forensic Genetics / 法医学杂志

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.

Progress of DNA-based Methods for Species Identification / 法医学杂志

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.

Forensic Investigation of Goldeneye™ DNA ID 22NC Kit / 法医学杂志

OBJECTIVE@#To investigate the genetic data of 21 autosomal STR included in Goldeneye™ DNA ID 22NC Kit in Chinese Han nationality and to evaluate the forensic application.@*METHODS@#By detected 500 unrelated healthy individuals in Chinese Han nationality of East China with Goldeneye™ DNA ID 22NC Kit, allele frequencies, population genetics parameters and linkage disequilibrium information of the 21 autosomal STR were statistically analyzed.@*RESULTS@#In the 21 autosomal STR, no deviations from Hardy-Weinberg equilibrium were detected and all loci were independent form each other. DP values of 21 autosomal STR were all above 0.85, and the combined discrimination power was 1-3.616 5 x 10(-26). Combined mean exclusion chance of this system in duo cases was 1-2.786 81 x10(-6), in trio cases was 1-8.545 82 x 10(-1).@*CONCLUSION@#Twenty-one autosomal STR included in Goldeneye™ DNA ID 22NC Kit are highly polymorphic in the Han nationality. Combined with Goldeneye™ DNA ID 20A Kit, the kit can satisfy the needs for full-sibling testing and facilitate the solution of this kind of case tools.

Development of an 18 X-InDel multiplex PCR system / 法医学杂志

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.

Forensic application of 30 InDel loci in Han and She nationalities of Eastern China / 法医学杂志

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.

Forensic validation of goldeneye? DNA ID 26Y system / 法医学杂志

OBJECTIVE@#To perform the validation and analysis of forensic parameters of Goldeneye DNA ID 26Y system.@*METHODS@#Based on the validation rules of Scientific Working Group on DNA Analysis Methods (SWGDAM), the kit was assessed from several parts, as test of PCR system, reproducibility, accuracy, and sensitivity, etc. And Y-STR loci of 517 unrelated healthy individuals from Eastern China were genotypes by this kit. The distribution and frequency of haplotype were calculated and forensic parameters of the kit were assessed.@*RESULTS@#The complete profiles can be obtained even when the PCR reaction volume with 6.25 microL. And correct profile was obtained with DNA down to 125 pg. No reproducible peaks were detected with the DNA of common animals and microorganism with the kit. For the male-male mixture testing, average 70% of the minor alleles were obtained when the ratios of 1:19 and 19:1. For the male-female mixture testing, results showed that the sensitivity of the kit was no compromised with the addition of female samples.@*CONCLUSION@#The validation studies demonstrated that Goldeneye DNA ID 26Y system has good sensitivity and specificity, and suitable for mixture testing. The polymorphism of 26 Y-STR loci included in this kit are good for forensic application.

Progress in InDel as a new generation of genetic marker / 法医学杂志

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.

Messenger RNA profiling for forensic body fluid identification: research and applications / 法医学杂志

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.

Typing and polymorphism analysis of 16 STR loci on X chromosome / 法医学杂志

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.

Algorithms of likelihood ratio for discriminating full sibling from half sibling / 法医学杂志

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.

Forensic application of investigator HDplex kit in Han nationality of Eastern China / 法医学杂志

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.

Screen and identification of causing-disease gene in a family with a special crystalline autosomal dominant congenital cataract / 中华实验眼科杂志

Background Inheritance is one of main causing-disease factors in congenital cataract.So the screen of causing-disease gene in congenital cataract patients is a critical step.Objective This survey was to investigate the molecular characteristics of a Chinese pedigree with a special crystalline autosomal dominant congenital cataract(ADCC) in Shanxi province.Methods This study was approved by Ethic Commission of Shanxi Eye Hospital.Informed consent was obtained from each subject before any medical examination.Twenty-two families from a pedigree with special crystalline were included in this study.The family members received regular ophthalmologic and general examinations to rule out any concomitant disorders.Blood samples were obtained to extract the DNA from all the subjects.Twenty-two fluorescent labeled microsatellites were selected from 17 causing genes of ADCC and amplified and screened for the linkage analysis.LOD was calculated and the candidate gene was directly sequenced.Results Ten individuals with congenital cataract were found in the family with the similar phenotype.The inheritance mode complied with the autosomal dominant pattern.Linkage analysis indicated a gene chain at D2S325 and D2S2358 with the LOD value 1.20(θ =0) and 0.22(θ =0).A known c.C70A(p.P23T) missence mutation at the coding region of CRYGD gene was detected by direct sequence.Conclusions A missense mutation P23T of the CRYGD gene cause the autosomal dominant congenital nuclear cataract with the special phenotype.

A primary study for criteria of full sibling determination with Identifiler system / 法医学杂志

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.

Genetic polymorphisms of 21 non-CODIS STR loci / 法医学杂志

OBJECTIVE@#To investigate genetic polymorphisms of 21 non-CODIS STR loci in Han population from the east of China and to explore their forensic application value.@*METHODS@#Twenty-one non-CODIS STR loci, were amplified with AGCU 21+1 STR kit and DNA samples were obtained from 225 unrelated individuals of the Han population from the east of China. The PCR products were analyzed with 3130 Genetic Analyzer and genotyped with GeneMapper ID v3.2 software. The genetic data were statistically analyzed with PowerStats v12.xls and Cervus 2.0 software.@*RESULTS@#The distributions of 21 non-CODIS STR loci satisfied the Hardy-Weinberg equilibration. The heterozygosity (H) distributions were 0.596-0.804, the discrimination power (DP) were 0.764-0.948, the probability of exclusion of duo-testing (PEduo) were 0.176-0.492, the probability of exclusion of trios-testing (PEtrio) were 0.334-0.663, and the polymorphic information content (PIC) were 0.522-0.807. The cumulative probability of exclusion (CPE) of duo-testing was 0.999707, the CPE of trios-testing was 0.9999994, and the cumulated discrimination power (CDP) was 0.99999999999999999994.@*CONCLUSION@#Twenty-one non-CODIS STR loci are highly polymorphic. They can be effectively used in personal identification and paternity testing in trios cases. They can also be used as supplement in the difficult cases of diad paternity testing.